Telomerase compositions and methods

ABSTRACT

Disclosed are various methods, compositions and screening assays connected with telomerase, including genes encoding the template RNA of  S. cerevisiae  telomerase and various telomerase-associated polypeptides.

The present application is a divisional application of U.S. applicationSer. No. 08/938,534, filed Sep. 26, 1997 now issued as U.S. Pat. No.5,916,752, and a divisional application of U.S. application Ser. No.08/431,080, filed Apr. 28, 1995 now issued as U.S, Pat. No. 5,698,686.is a continuation-in-part of U.S. patent application Ser. No.08/326,781, filed Oct. 20, 1994, now abandoned the entire text andfigures of which disclosure is specifically incorporated herein byreference without disclaimer.

The U.S. Government owns rights in the present invention pursuant toNational Institutes of Health Grants GM43893, GM07281 and CA14599 andArmy Research Office Grant DAAH04-93-G-0274.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates to telomerase compositions and methodsconnected therewith. Particularly disclosed are genes encoding thetemplate RNA of telomerase in Saccharomyces cerevisiae and varioustelomerase-associated proteins. Methods of using such genes and otherrelated biological components are also provided.

B. Description of the Related Art

DNA polymerases synthesize DNA in a 5′ to 3′ direction and require aprimer to initiate synthesis. These restrictions pose a problem for thecomplete replication of linear chromosomes (Watson, 1972; Olovnikov,1973). In the absence of a specialized mechanism to maintain terminalsequences, multiple replication cycles would cause chromosomes toprogressively shorten from their ends.

Telomeres are specialized nucleoprotein complexes that constitute theends of eukaryotic chromosomes and protect them from degradation andend-to-end fusion (Zakian, 1989; Blackburn, 1991; Price, 1991; Henderson& Larson, 1991; Wright et al., 1992; Blackburn, 1994). When telomeresare absent, the instability of non-telomeric chromosomal ends leads tochromosome loss (Sandell & Zakian, 1993). In addition, telomeres arerequired for the complete replication of chromosomes (Zakian, 1989;Blackburn, 1991; Price, 1991; Henderson & Larson, 1991; Wright et al.,1992; Blackburn, 1993; 1994).

In many eukaryotes, telomeres are composed of simple tandem repeats,with the 3′-terminal strand composed of G-rich sequences (Zakian, 1989;Blackburn, 1991; Price, 1991; Henderson & Larson, 1991; Wright et al.,1992; Blackburn, 1994). Certain insights into the mechanism by whichtelomeric DNA is maintained has come from the identification oftelomerase activity in several species of ciliates, as well as inextracts of Xenopus, mouse, and human cells (Greider & Blackburn, 1985;1987; 1989; Zahler & Prescott, 1988; Morin, 1989; Prowse et al., 1993;Shippen-Lentz & Blackburn, 1989; Mantell & Greider, 1994).

Telomerase is a ribonucleoprotein enzyme that elongates the G-richstrand of chromosomal termini by adding telomeric repeats (Blackburn,1993). This elongation occurs by reverse transcription of a part of thetelomerase RNA component, which contains a sequence complementary to thetelomere repeat. Following telomerase-catalyzed extension of the G-richstrand, the complementary DNA strand of the telomere is presumablyreplicated by more conventional means.

Germline cells, whose chromosomal ends must be maintained throughrepeated rounds of DNA replication, do not decrease their telomerelength with time, presumably due to the activity of telomerase (Allsoppet al., 1992). In contrast, somatic cells appear to lack telomerase, andtheir telomeres shorten with multiple cell divisions (Allsopp et al.,1992; Harley et al., 1990; Hastie et al., 1990; Lindsey et al., 1991;Vaziri et al., 1993; Counter et al., 1992; Shay et al., 1993;Klingelhutz et al., 1994; Counter et al., 1994a;b).

Telomerase is believed to have a role in the process of cell senescence(de Lange, 1994; Greider, 1994; Harley et al., 1992). The repression oftelomerase activity in somatic cells is likely to be important incontrolling the number of times they divide. Indeed, the length oftelomeres in primary fibroblasts correlates well with the number ofdivisions these cells can undergo before they senescence (Allsopp etal., 1992). The loss of telomeric DNA may signal to the cell the end ofits replicative potential, as part of an overall mechanism by whichmulticellular organisms limit the proliferation of their cells.

Due to its role in controlling replication, telomerase has also recentlybeen implicated in oncogenesis (de Lange, 1994; Greider, 1994; Harley etal., 1992). It is thought that late stage tumors probably require thereactivation of telomerase in order to avoid total loss of theirtelomeres and massive destabilization of their chromosomes. Immortalizedcell lines produced from virally transformed cultures have activetelomerase and stable telomere lengths (Counter et al., 1992; Shay etal., 1993; Klingelhutz et al., 1994; Counter et al., 1994b). Recently,telomerase activity has also been detected in human ovarian carcinomacells (Counter et al., 1994a).

Telomerase is thus an important component of eukaryotic cells, thedysfunction of which can have significant consequences. Although presentknowledge concerning telomerase is increasing, there is a marked needfor individual telomerase components to be isolated and for furtheranalytical methods to be developed. The creation of a system formanipulating telomerase in a genetically tractable eukaryotic organismwould be particularly valuable.

SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks inherent inthe prior art by providing purified telomerase components and systemsfor isolating further components and for developing agents with thecapacity to modify telomerase actions. Particular aspects of-thisinvention concern the isolation and uses of severaltelomerase-associated genes from Saccharomyces cerevisiae, including thetelomerase RNA template gene.

In certain aspects, this invention concerns nucleic acid segments thathybridize to, or that have sequences in accordance with, SEQ ID NO:1,SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23.SEQ ID NO:1 represents a telomerase RNA template-encoding sequence, alsotermed TLC1; and each of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQID NO:31 and SEQ ID NO:23 represent sequences that encodetelomerase-associated polypeptides, also termed STR sequences (STR1,STR3, STR4, STR5 and STR6, respectively).

Both the gene TLC1 (SEQ ID NO:1 and the complementary sequence, SEQ IDNO:4), and the template RNA, include a CA-rich region. The CA-richregion is represented by SEQ ID NO:3. In the RNA template, the CA-richregion is reversed transcribed to synthesize the GT-rich telomericrepeats. An example of the GT-rich telomeric sequence is represented bySEQ ID NO:2.

The present invention generally concerns non-ciliate eukaryotictelomerase components. These are represented by telomerase componentsfrom mammalian cells, including human cells, and telomerase componentsfrom other non-ciliate species. One significant contribution of thisinvention is the development of methods of utilizing telomerasecomponents, which methods are functional in useful eukaryotic cells.“Useful eukaryotic cells” particularly include human cells, as these aredirectly relevant to the development of diagnostics and therapeutics forhuman use, and cells of genetically tractable eukaryotic organisms, asthese are recognized to have significant value in scientific terms and,ultimately, in drug development. The preferred non-ciliate telomerasecomponents of the invention are thus mammalian, drosophila and yeasttelomerase components.

A. DNA Segments and Vectors

The invention thus provides nucleic acid segments that are characterizedas nucleic acid segments that include a sequence region that consists ofat least 17 contiguous nucleotides that have the same sequence as, orare complementary to, 17 contiguous nucleotides of SEQ ID NO:1, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23.

The nucleic acid segments of the invention are further characterized asbeing of from 17 to about 10,000 nucleotides in length, which nucleicacid segments hybridize to the nucleic acid segment of SEQ ID NO:1, SEQID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, orthe complement thereof, under standard hybridization conditions.

“Complementary” or “complement”, in terms of nucleic acid segments thatare complementary to those listed above, or that hybridize to acomplement of such nucleic acid segments, means that the nucleic acidsequences are capable of base-pairing to a given sequence, such as thesequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQID NO:31 or SEQ ID NO:23, according to the standard Watson-Crickcomplementarity rules. That is, the larger purines will base pair withthe smaller pyrimidines to form combinations of guanine paired withcytosine (G:C) and adenine paired with either thymine (A:T), in the caseof DNA, or adenine paired with uracil (A:U) in the case of RNA.

Encompassed within the nucleic acid sequences of the invention arefull-length DNA sequences or other DNA segments that have a sequenceregion that encodes a peptide, polypeptide or protein and that may beused, for example, in recombinant expression. Also included within thenucleic acid sequences are DNA and RNA segments for use in nucleic acidhybridization embodiments, such as in cloning.

The smaller nucleic acid segments may be termed probes and primers. Theindividual sequences of 17, 20, 30, 50 or so nucleotide sequencestretches, for example, may be readily identified by “breaking down” thelonger sequences disclosed herein to provide one or more shortersequences. Using an exemplary length of 17 bases, each of the 17-merpossibilities from the DNA sequences described herein have been definedand are listed in Table 2.

In certain embodiments, the invention provides isolated DNA segments andrecombinant vectors that have one or more sequence regions that encodeone or more non-ciliate eukaryotic telomerase components, andpreferably, those that encode one or more yeast (S. cerevisiae)telomerase components. The creation and use of recombinant host cells,through the application of DNA technology, that express yeast or othernon-ciliate eukaryotic telomerase components is also encompassed by theinvention.

As used herein, the term “telomerase component” refers to a biologicalcomponent that is associated with a non-ciliate eukaryotic telomerasecomplex, such as a mammalian, drosophila or yeast telomerase component.Preferably, the telomerase components will be associated with a yeasttelomerase complex. A “telomerase complex” in this sense is aribonucleoprotein enzyme complex that functions to elongate the G-richstrand of eukaryotic, and preferably yeast, chromosomal termini byadding telomeric repeats. Telomerase components (ortelomerase-associated components) therefore include both RNA andpolypeptidyl components.

An important component of telomerase is the telomerase RNA template ortemplate sequence. The term “telomerase RNA template”, as used herein,refers to a non-ciliate eukaryotic, such as a mammalian, drosophila, orpreferably, a yeast telomerase RNA component that includes a sequencethat is complementary to the telomere repeat, i.e., that iscomplementary to the G-rich or GT-rich sequences of chromosomal termini.The telomerase RNA template is thus an isolated RNA component that has aC-rich or CA-rich sequence and that, by interacting with othertelomerase components, functions to extend telomeric repeats. Thetelomerase RNA template may also be defined as the telomerase substratefor reverse transcription.

Further telomerase components are telomerase-associated proteins andpolypeptides. The “telomerase-associated proteins and polypeptides” ofthis invention are proteins, polypeptides or peptides that are requiredfor telomerase function in non-ciliate eukaryotic cells, and preferably,in yeast cells. Such telomerase-associated proteins and polypeptideswill generally be physically and functionally associated with thetelomerase complex in the nucleus, however, they may also be proteins orpolypeptides that only associate with the telomerase complex for certainperiods of time, at defined points of the cell cycle, or may be presentonly in certain cell types of a multicellular organism.

Telomerase-associated proteins, polypeptides and peptides may haveeither functional or structural roles within the telomerase complex.That is, they may have a catalytic or regulatory role, or may form thescaffolding of the telomerase structure. The telomerase-associatedproteins or polypeptides may function only in terms of telomeraseactivity, i.e., they may be telomerase-restricted; or they may haveother biological functions within the cell nucleus, such as in otheraspects of chromosome replication and stability, or may even havecytoplasmic functions.

The telomerase DNA segments of the present invention are thus DNAsegments isolatable from non-ciliate eukaryotic cells, and preferably,from yeast cells, that are free from total genomic DNA and that includea sequence region that is capable of expressing a telomerase RNA orpolypeptide component. The DNA segments may, in certain embodiments,also be defined as those capable of inhibiting the telomerase activityof a cell by over-expression in a cell that previously containedtelomerase activity. In further embodiments, the DNA segments may bedefined as those capable of conferring telomerase activity to a hostcell when incorporated into a cell that has been rendered deficient insuch activity.

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species, such asa mammal, drosophila or yeast species. Therefore, a DNA segment thatcomprises a sequence region that encodes a telomerase-associatedcomponent refers to a DNA segment that includes telomerase-associatedcomponent coding sequences or regions, yet is isolated away from, orpurified free from, total genomic DNA of the species from which the DNAsegment is obtained. Included within the term “DNA segment”, are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

Similarly, a telomerase-associated gene is a DNA segment comprising anisolated or purified gene that includes a sequence region that encodes acomponent associated with a mammalian, drosophila, or preferably, with ayeast telomerase. The term “an isolated gene associated with anon-ciliate eukaryotic telomerase”, as used herein, refers to a DNAsegment including telomerase RNA or protein coding sequences or regionsand, in certain aspects, regulatory sequences, isolated substantiallyaway from other naturally occurring genes or encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalRNA, protein, polypeptide or peptide encoding unit or region. As will beunderstood by those in the art, this functional term includes bothgenomic sequences, cDNA sequences and smaller engineered gene segmentsthat express, or may be adapted to express, RNA molecules, proteins,polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case a telomerase-associated gene, forms thesignificant part of the sequence or coding region of the DNA segment,and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or cDNA coding regions. Of course, this refers tothe DNA segment as originally isolated, and does not exclude genes orcoding regions later added to the segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that include anisolated gene or sequence region that encodes a non-ciliate eukaryotictelomerase RNA template, such as a mammalian, drosophila, or preferably,a yeast telomerase RNA template. This aspect of the invention isexemplified by DNA segments and genes that encode the S. cerevisiaetelomerase RNA template sequence of CACCACACCCACACAC (SEQ ID NO:3).

A variety of oligonucleotides, DNA segments and genes that encode theCACCACACCCACACAC (SEQ ID NO:3) telomerase RNA template sequence are madepossible by the discovery of the present inventors'. These includesequences from SEQ ID NO:1, and the complementary strand, SEQ ID NO:4.The sequence from SEQ ID NO:1 that includes the template-encoding regionof CACCACACCCACACAC (SEQ ID NO:3) is particularly represented by thecontiguous DNA sequence from position 468 to position 483 of SEQ IDNO:1. Such DNA segments will have a minimum length of 17 nucleotides,and are exemplified by the contiguous DNA sequences from position 467 toposition 483, or from position 468 to position 484, of SEQ ID NO:1.

DNA segments longer than 17 bases are also contemplated, in incrementsof single integers up to and including the 1301 bases of SEQ ID NO:1,and even longer. The contiguous sequences from SEQ ID NO:1 may beequidistant around the template-encoding region of SEQ ID NO:3, or theymay have the SEQ ID NO:3 region located substantially towards thebeginning or towards the end of the given sequence. DNA segments maythus have sequences in accordance with the contiguous sequences betweenabout position 450 or 460 and about position 485 of SEQ ID NO:1; betweenabout position 300 or 400 and about position 500, 600 or 700 of SEQ IDNo:1; between about position 100 or 200 and about position 800, 900,1000, 1100 or 1200 of SEQ ID NO:1; or between any of the afore-mentionedranges and intermediates thereof. DNA segments and isolated genes thatinclude the full-length DNA sequence of SEQ ID NO:1 are alsocontemplated.

In further embodiments, the invention provides isolated DNA segments,genes and vectors incorporating DNA sequences that encode a non-ciliateeukaryotic telomerase-associated polypeptide, such as a mammalian,drosophila or yeast, telomerase-associated polypeptide, as exemplifiedby yeast polypeptides that includes within their amino acid sequence acontiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 or SEQ ID NO:24.

The term “a contiguous amino acid sequence from SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24” means that acontiguous sequence is present that substantially corresponds to acontiguous portion of one of the afore-mentioned sequences and hasrelatively few amino acids that are not identical to, or a biologicallyfunctional equivalent of, the amino acids of SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24. The term “biologicallyfunctional equivalent” is well understood in the art and is furtherdefined in detail herein. Accordingly, sequences that have between about70% and about 80%; or more preferably, between about 81% and about 90%;or even more preferably, between about 91% and about 99%; of amino acidsthat are identical or functionally equivalent to the amino acids of SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 willbe sequences in accordance with the present invention.

The protein-encoding DNA segments, genes and vectors may include withintheir sequence region a contiguous nucleic acid sequence from SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. Theterm “a contiguous nucleic acid sequence from SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23” is used in the samesense as described above and means that the nucleic acid sequencesubstantially corresponds to a contiguous portion of one of thedesignated sequences and has relatively few codons that are notidentical, or functionally equivalent, to the codons of SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids, as is known in the art and further described herein (seeTable 1).

Protein-encoding DNA segments and genes of the present invention mayencode a full length telomerase-associated protein or polypeptide, asmay be used in expressing the protein. Such DNA segments are exemplifiedby those that comprise an isolated gene that includes a contiguous DNAsequence substantially as shown between position 54 and position 1799 ofSEQ ID NO:29, that encodes a polypeptide substantially as shown in SEQID NO:16; or that includes a contiguous DNA sequence substantially asshown between position 78 and position 1094 of SEQ ID NO:30, thatencodes a polypeptide substantially as shown in SEQ ID NO:18; or thatincludes a contiguous DNA sequence substantially as shown betweenposition 2 and position 2368 of SEQ ID NO:19, that encodes a polypeptidesubstantially as shown in SEQ ID NO:20; or that includes a contiguousDNA sequence substantially as shown between position 55 and position 699of SEQ ID NO:31, that encodes a polypeptide substantially as shown inSEQ ID NO:22; or that includes a contiguous DNA sequence substantiallyas shown between position 3 and position 1955 of SEQ ID NO:23, thatencodes a polypeptide substantially as shown in SEQ ID NO:24.

For both protein expression and hybridization, the nucleic acid segmentsused may include the full length versions of any of thetelomerase-associated genes disclosed herein, or their biologicalequivalents, including their complementary sequences where hybridizationis concerned. This is exemplified by DNA segments that have, or thatcomprise a sequence region that has, the 1301 nucleotides of SEQ IDNO:1, the 1882 nucleotides of SEQ ID NO:29, the 1094 nucleotides of SEQID NO:30, the 2434 nucleotides of SEQ ID NO:19, the 807 nucleotides ofSEQ ID NO:31, the 2117 nucleotides of SEQ ID NO:23, or any substantiallyequivalent sequences.

Further, the present DNA segments may be used to express proteinfragments or peptides, for example, peptides of from about 15 to about30, about 50 or about 100 amino acids in length. The peptides may, ofcourse, be of any length between or around such stated ranges, with“about” meaning a range of lengths in positive integers between eachabove-listed reference point and higher, with 12-15 or so being theminimum length. Appropriate coding sequences and regions may be readilyidentified from any of the regions of SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:19, SEQ ID NO:31 or SEQ ID NO:23.

The sequence or coding regions of the invention will be a minimum lengthof 17 nucleotides, and will most often be longer than this, such asupwards of about 40-50 nucleotides in length or so. The maximum lengthof the DNA segments is not limited by the length of the coding regionsthemselves, so that DNA segments of about 1,000, about 3,000, about5,000 and 10,000 or even longer are contemplated. It will be readilyunderstood that all lengths intermediate between the above-quoted rangesare also included.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be substantially asshown in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above. The addition of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region or may include various internal sequences,i.e., introns, which are known to occur within genes.

Excepting intronic or flanking regions, and allowing for the degeneracyof the genetic code, sequences that have between about 70% and about80%; or more preferably, between about 80% and about 90%; or even morepreferably, between about 90% and about 99% of nucleotides that areidentical to the nucleotides of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23 will be sequences that aresubstantially as shown in such sequences. From the inventors'experience, sequences with 70% identity or higher are expected to betelomerase-related sequences.

The nucleic acid segments of the present invention, regardless of thelength of any coding sequences themselves, may be combined with othernucleic acid sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol.

As stated above, the invention is not limited to the particularsequences of SEQ ID NO:1, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQID NO:31 or SEQ ID NO:23 (nucleic acid), or SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24 (amino acid). In terms ofexpression, recombinant vectors may therefore variously include thetelomerase-associated protein coding regions themselves, coding regionsbearing selected alterations or modifications in the basic codingregion, or they may encode larger polypeptides that nevertheless includesuch telomerase-associated protein coding regions or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

For protein expression embodiments, the DNA segments may includebiologically functional equivalent protein-coding sequences that havearisen as a consequence of codon redundancy and functional equivalency,as is known to occur naturally within biological sequences.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein or to test telomerase mutants in orderto examine their activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the telomerase-associated protein coding regions are alignedwithin the same expression unit with other proteins or peptides havingdesired functions, such as for purification or immunodetection purposes(e.g., proteins that may be purified by affinity chromatography andenzyme label coding regions, respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich an RNA or protein coding portion of a DNA segment, whetherencoding an RNA template, a full length protein or smaller peptide, ispositioned under the control of a promoter. The promoter may be in theform of the promoter that is naturally linked to a telomerase-associatedgene, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR technology, in connection with thecompositions disclosed herein.

In other expression embodiments, it is contemplated that certainadvantages will be gained by positioning a coding DNA segment orsequence region under the control of a recombinant, or heterologous,promoter. As used herein, a recombinant or heterologous promoter isintended to refer to a promoter that is not normally associated with atelomerase-associated gene in its natural environment. Such promotersmay include yeast promoters normally associated with other genes, and/orpromoters isolated from any other bacterial, viral, insect or mammaliancell.

Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al.(1989). The promoters employed may be constitutive, or inducible, andcan be used under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides.

Preferred promoter systems contemplated for use in high-level expressionin S. cerevisiae include, but are not limited to, the GAL1, MET3, andPGK promoter systems. For conditional alleles, as may be used incellular studies of the RNA template, a chimeric fusion of an RNAtemplate gene may be placed under the regulation of a heterologouspromoter. Appropriate promoters include the MET3 promoter, which isrepressed in the presence of methionine and induced when methionine isabsent from the medium; and the GAL1,10 UAS, as described in Example XI.

B. Nucleic Acid Hybridization

In addition to their use in directing the expression oftelomerase-associated RNA and protein components, the nucleic acidsequences disclosed herein also have a variety of other uses, forexample, in nucleic acid hybridization embodiments. The ability ofnucleic acid probes or primers to specifically hybridize to thetelomerase-associated nucleic acid sequences disclosed herein willenable them to be of use in detecting the presence of complementarysequences in a given sample. However, other uses are envisioned,including the use of the sequence information for the preparation ofmutant species primers, or primers for use in preparing other geneticconstructs.

The present invention thus concerns nucleic acid segments of 17nucleotides in length, or longer, that hybridize to thetelomerase-associated sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or their complements,under standard hybridization conditions. This provides another physicaland functional definition for identifying additional sequences inaccordance with the invention, as well as defining useful sub-sequences,such as primers.

The nucleic acids that hybridize to the sequences of SEQ ID NO:1, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, may be17 nucleotides in length or longer, such as about 20, about 25, about30, about 50, about 75, about 100, about 150, about 200, about 250,about 500, about 750 or about 1,000 nucleotides in length, or evenlonger. As the length of the nucleic acid segment that hybridizes is notsolely a function of the length of the substantially complementarysequence region, these nucleic acid segments may also be about 2,000,about 3,000, about 5,000 or about 10,000 nucleotides in length orlonger, so long as the total length does not prevent hybridization underthe conditions defined herein.

As with the sequence or coding regions defined hereinabove, it will bereadily understood that any intermediate length between the quotedranges is included, such as 17, 18, 19, 20, 21, 22, 23, etc; 50, 51, 52,53, etc.; 100, 101, 102, 103, etc.; including all positive integersthrough the 150-500; 500-1,000; 1,000-2,000; 2,000-5,000; and5,000-10,000 ranges, up to and including sequences of about 12,001,12,002, 13,001, 13,002 and the like.

The total size of nucleic acid segment or fragment, as well as the sizeof the complementary stretch(es), will ultimately depend on the intendeduse or application of the particular nucleic acid segment. The use of ahybridization probe of about 17 nucleotides in length allows theformation of a duplex molecule that is both stable and selective.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of telomerase-associated genes or cDNAs. Depending on theapplication envisioned, one will desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence.

For applications requiring high selectivity, one will typically desireto employ relatively stringent conditions to form the hybrids, e.g., onewill select relatively low salt and/or high temperature conditions thattolerate little, if any, mismatch between the probe and the template ortarget strand. Standard high stringency hybridization conditions aredescribed in the hybridization protocols set forth herein in thedetailed description.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, or where one seeks to isolate telomerase-associatedsequences from related species, functional equivalents, or the like,less stringent hybridization conditions are useful to allow formation ofthe heteroduplex. In these circumstances, one may desire to employstandard low stringency hybridization conditions, which are alsodescribed in the hybridization protocols set forth in the detaileddescription.

Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex, in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

Where hybridization probes or primers are to be designed from aconsideration of the longer sequences disclosed herein, they may beselected from any portion of any of the nucleic acid sequences. All thatis required is to review the sequences and to select any continuousportion of the sequence, from 17 nucleotides in length up to andincluding the full length sequence.

Once the coding sequence of a telomerase-associated gene has beendetermined, various primers can be designed around that sequence.Primers may be of any length, but typically, are 17, 20, 25 or 30 basesor so in length. By assigning numeric values to a sequence, for example,the first residue is 1, the second residue is 2, and the like, analgorithm defining all primers is:

n to n+y

where n is an integer from 1 to the last number of the sequence and y isthe length of the primer minus one, in the above cases (16, 19, 24, 29),where n+y does not exceed the last number of the sequence. For example,for the TLC1 gene, n is 1 to 1301. Thus, for a 17-mer, the probescorrespond to bases 1 to 17, 2 to 18, 3 to 19 . . . up to 1285 to 1301.Table 2 herein sets forth the number of contiguous 17-mer sequences thatmay be obtained from the sequences of SEQ ID NO:1, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23, or their complements.

The choice of probe and primer sequences may be governed by variousfactors, such as, by way of example only, one may wish to employ primersfrom towards the termini of the total sequence, or from the ends of anyfunctional domain-encoding sequences, in order to amplify further DNA;one may employ probes corresponding to the entire DNA, or to the RNAtemplate region, to clone template genes from other species or to clonefurther telomerase template-like or homologous genes from any speciesincluding human; one may also design appropriate probes or primers toscreen biological samples to identify cells with inappropriatetelomerase levels or activity, as may be related to cancer or even toinfertility.

The process of selecting and preparing a nucleic acid segment thatincludes a contiguous sequence from within SEQ ID NO:1, SEQ ID NO:29,SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ ID NO:23 may be readilyachieved by, for example, directly synthesizing the fragment by chemicalmeans, as is commonly practiced using an automated oligonucleotidesynthesizer. Also, fragments may be obtained by application of nucleicacid reproduction technology, such as the PCR technology of U.S. Pat.No. 4,683,202 and U.S. Pat. No. 4,682,195 (each incorporated herein byreference), by introducing selected sequences into recombinant vectorsfor recombinant production, and by other recombinant DNA techniquesgenerally known to those of skill in the art of molecular biology. Ofcourse, smaller nucleic acid fragments may also be obtained by othertechniques such as, e.g., by mechanical shearing or by restrictionenzyme digestion.

In certain embodiments, it will often be advantageous to employ nucleicacid sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of giving a detectable signal. Incertain embodiments, one will likely desire to employ a fluorescentlabel or an enzyme tag, such as urease, alkaline phosphatase orperoxidase, instead of radioactive or other environmental undesirablereagents. In the case of enzyme tags, colorimetric indicator substratesare known that can be employed to provide a means visible to the humaneye or spectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

C. Further Telomerase Compositions

The present invention further includes isolated RNA segments, of from 17to about 1,500 nucleotides in length, that comprise a non-ciliate, orpreferably, a yeast telomerase RNA template. The isolated RNA segmentswill be obtained free from total nucleic acids, chromosomes and intacttelomerase complexes, and will include a non-ciliate eukaryotic, orpreferably, a yeast telomerase RNA template. This is exemplified by RNAsegments including the S. cerevisiae RNA template sequence ofCACCACACCCACACAC (SEQ ID NO:3).

Isolated RNA segments that include the minimum functional mammalian,drosophila or yeast telomerase RNA template coding sequences and theminimum upstream sequences necessary for expression are alsocontemplated. These may be identified as described herein in Example XIand will be useful in mutant analysis, promoter and expression analysisand creation of conditional mutants.

Isolated RNA segments that have substantially the same secondarystructure as the RNA segment encoded by the sequence of SEQ ID NO:1 arealso included within the scope of the present invention. This may beassessed by techniques, and computer programs, that predict secondarystructure based on the primary sequence of the RNA. The secondarystructure predictions are supported by mutant/function analysis, as iswell known in the art. That is, given the predicted structure, it isstraightforward for the ordinary artisan to accurately predict theeffects of certain sets of mutations in the RNA.

Further compositions in accordance with this invention include affinitycolumns that comprise a deoxyoligonucleotide attached to a solidsupport, where the deoxyoligonucleotide includes a sequencecomplementary to a non-ciliate, or preferably, a yeast telomerase RNAtemplate sequence. Such deoxyoligonucleotides and affinity columns willbe capable of binding eukaryotic, or preferably, yeast telomerasecomplexes, enabling their purification. As the template RNA includes theCA-rich template region, an appropriate column-bound bait will be aGT-rich DNA sequence, as represented, by way of example only, by SEQ IDNO:2.

The oligonucleotides may be attached to any one of a variety of solidsupports for use in standard column chromatography or in FPLC or HPLCtechniques. Oligonucleotides may be attached using a variety ofappropriate methods, such as, by way of example, using direct chemicalconjugation, or other means such as biotin-avidin linkers, and the like.All such techniques are routine in the art.

Still further embodiments of the present invention concern recombinanthost cells that contain or incorporate a DNA segment or recombinantvector that comprises an isolated gene associated with non-ciliateeukaryotic, or preferably, with yeast telomerase. Thetelomerase-associated components, whether they be cDNA or genomic, maybe used in expression systems for the recombinant preparation of RNAtemplates or telomerase-associated polypeptides.

As used herein, the term “engineered” or “recombinant” cell is intendedto refer to a cell into which an exogenous DNA segment or gene, such asa cDNA or gene encoding a telomerase-associated component has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occurring cells that do not contain a recombinantly introducedexogenous DNA segment or gene. Engineered cells are thus cells having agene or genes introduced through the hand of man. Recombinantlyintroduced genes will either be in the form of a cDNA gene (i.e., theywill not contain introns), a copy of a genomic gene, or will includegenes positioned adjacent to a promoter not naturally associated withthe particular introduced gene.

The engineering of DNA segment(s) for expression in prokaryotic oreukaryotic systems is performed using techniques known to those of skillin the art, and further described herein in detail. It is believed thatvirtually any prokaryotic or eukaryotic host cell system may be employedin the expression of one or more telomerase-associated components, withyeast systems being preferred in certain embodiments.Telomerase-associated polypeptides may also be as fusions with, e.g.,β-galactosidase, ubiquitin, Schistosoma japonicum glutathioneS-transferase, and the like.

To achieve expression, one would position the telomerase codingsequences adjacent to and under the control of a promoter. It isunderstood in the art that to bring a coding sequence under the controlof such a promoter, one positions the 5′ end of the transcriptioninitiation site of the transcriptional reading frame of the proteinbetween about 1 and about 50 nucleotides or so “downstream” of (i.e., 3′of) the chosen promoter.

Where eukaryotic expression is contemplated, one will also typicallydesire to incorporate into the transcriptional unit which includes theenzyme, an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

Generally speaking, it may be more convenient to employ as therecombinant gene a cDNA version of the gene. It is believed that the useof a cDNA version will provide advantages in that the size of the genewill generally be much smaller and more readily employed to transfectthe targeted cell than will a genomic gene, which will typically be upto an order of magnitude larger than the cDNA gene. However, theinventors do not exclude the possibility of employing a genomic versionof a particular gene where desired.

The recombinant host cells of the invention will effectively expresses aDNA segment to produce a telomerase RNA template or a polypeptideassociated with telomerase. The invention thus further includesrecombinant gene products that are prepared by expressing a eukaryotic,or preferably, a yeast telomerase-associated gene in a recombinant hostcell and purifying the expressed gene product away from totalrecombinant host cell components. The gene products include telomeraseRNA templates, proteins, polypeptides and peptides associated withtelomerase, and combinations and equivalents thereof.

The preparation of such recombinant gene products is preferably achievedby using a DNA segment of the invention in the preparation of arecombinant vector in which a telomerase-associated gene is positionedunder the control of a promoter. The recombinant vector is thenintroduced into a recombinant host cell, which is cultured underconditions effective, and for a period of time sufficient, to allowexpression of the telomerase-associated gene, which thus allows theexpressed gene product to be collected, giving a purified preparation.

The invention further concerns recombinant RNA segments that includenon-ciliate telomerase RNA templates, such as mammalian, drosophila, orpreferably, yeast telomerase RNA templates; and

recombinant protein and polypeptide compositions, free from total cellcomponents, that comprise one or more purified non-ciliate, orpreferably, yeast telomerase-associated components. These areexemplified by polypeptides that include a contiguous amino acidsequence from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 orSEQ ID NO:24.

The terms “purified telomerase-associated polypeptide and RNA template”,as used herein, refer to telomerase-associated polypeptide or RNAtemplate compositions, isolatable from eukaryotic, or preferably, fromyeast cells, wherein the polypeptide or RNA is purified to any degreerelative to its naturally-obtainable state, e.g., relative to its puritywithin a cellular extract. More preferably, “purified” refers totelomerase-associated polypeptide or RNA template compositions that havebeen subjected to fractionation to remove various non-telomerasecomponents. “Substantially purified” native and recombinant telomeraseRNA templates and polypeptides are also preparable using the methods ofthe invention.

To prepare a purified telomerase-associated component in accordance withthe present invention one would subject a composition to fractionationto remove various non-telomerase-associated components. Varioustechniques suitable for use in RNA and protein purification will be wellknown to those of skill in the art. Protein purification techniquesinclude, for example, precipitation with ammonium sulphate, PEG,antibodies, and the like, or by heat denaturation, followed bycentrifugation; chromatography steps such as ion exchange, gelfiltration, reverse phase, hydroxylapatite and affinity chromatography;isoelectric focusing; gel electrophoresis; and combinations of such andother techniques.

Specific examples of purification schemes for use in the presentinvention are those including initial separation of nuclear proteins,followed by gradient centrifugation methods (equilibrium andsedimentation velocity), column chromatography and gel electrophoresis,as described in Example XV. Specific binding to RNA or DNA segmentsrelated to the telomerase template sequences, including affinity columnbinding embodiments, is also envisioned to be particularly useful.

For assays of intact or relatively intact telomerase complexes,deoxyoligonucleotide substrates, representing 3′ G-rich telomere tails,are incubated in cellular extracts containing telomerase with³²P-labeled dNTP's (typically dGTP or dTTP). The products of telomeraseelongation on the input deoxyoligonucleotide substrate may then bedetected by, e.g., gel electrophoresis and autoradiography. A series ofsubstrates is also preferably used, as described in Example XV.

Although preferred for use in certain embodiments, there is no generalrequirement that the RNA or proteins always be provided in their mostpurified state. Indeed, it is contemplated that less substantiallypurified telomerase-associated components, which are nonethelessenriched relative to their natural state, will have utility in certainembodiments. These include, for example, certain binding assays,screening protocols, titration of components, and the like. Inactiveprotein fractions also have utility, for example, in antibodygeneration.

In further embodiments, the invention also provides polyclonal ormonoclonal antibodies that bind to a non-ciliate, and preferably, to ayeast telomerase-associated polypeptide, as exemplified by an antibodythat has binding affinity for a protein or peptide that includes acontiguous amino acid sequence from SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 or SEQ ID NO:24. Cross-reactive antibodies are alsoencompassed by the invention, as may be identified by employing acompetition binding assay, such as an ELISA or RIA, as are well known inthe art.

Particular techniques for preparing antibodies in accordance with theinvention are disclosed herein, which methods generally compriseadministering to an animal a composition comprising an immunologicallyeffective amount of a telomerase-associated component protein, peptideor other epitopic composition. By “immunologically effective amount” ismeant an amount of a telomerase-associated protein or peptidecomposition that is capable of generating an immune response in therecipient animal, and particularly, in this case, generating an antibodyor B cell response.

Any of the DNA, RNA, proteins, polypeptides and antibodies of thisinvention may also be linked to a detectable label, such as aradioactive, fluorogenic, biological, chromogenic or even a nuclearmagnetic spin resonance label. Biolabels such as biotin and enzymes thatare capable of generating a colored product upon contact with achromogenic substrate will be preferred in certain embodiments.Exemplary enzyme labels include alkaline phosphatase, hydrogenperoxidase, urease and glucose oxidase enzymes.

In still further embodiments, the invention concerns molecularbiological and immunodetection kits. The labelled nucleic acid segments,proteins or antibodies may be employed to detect othertelomerase-associated nucleic acid, protein or antibody components inextracts, cells or biological samples, as may be used in the detectionof telomerase in clinical samples, or in the purification oftelomerase-associated components, as appropriate. The kits willgenerally include a suitable telomerase-associated nucleic acid segmentor antibody together with an detection reagent, and a means forcontaining the telomerase-associated component and reagent.

The detection reagent will typically comprise a label associated withthe telomerase nucleic acid segment or antibody, or even associated witha secondary binding ligand. Exemplary ligands include secondaryantibodies directed against a first antibody. The kits may containtelomerase-associated nucleic acid segments or antibodies either infully conjugated form, in the form of intermediates, or as separatemoieties to be conjugated by the user of the kit.

Kits for use in molecular biological tests to identifytelomerase-associated components may also contain one or more unrelatednucleic acid probes or primers for use as controls, and optionally, oneor more further molecular biological reagents, such as restrictionenzymes or PCR components. The components of the kits will preferably bepackaged within distinct containers.

The container means for any of the kits will generally include at leastone vial, test tube, flask, bottle, syringe or other container means,into which the nucleic acid or antibody may be placed, and preferablysuitably aliquoted. Where a second component, e.g., a binding ligand isprovided, the kit will also generally contain a second vial or othercontainer into which this ligand or antibody may be placed. The kits ofthe present invention will also typically include a means for containingthe vials in close confinement for commercial sale, such as, e.g.,injection or blow-molded plastic containers into which the desired vialsare retained.

D. Telomerase-Associated Methods

The invention further provides methods for detecting non-ciliateeukaryotic, and preferably, yeast telomerase-associated genes or nucleicacid segments in samples, such as cells, cellular extracts, partiallypurified telomerase compositions and other biological and even clinicalsamples. Such methods generally comprise obtaining sample nucleic acidsfrom a sample suspected of containing a telomerase-associated gene;contacting the sample nucleic acids with a telomerase-associated nucleicacid segment as described herein under conditions effective to allowhybridization of substantially complementary nucleic acids; anddetecting the hybridized complementary nucleic acids thus formed.

A variety of hybridization techniques and systems are known that can beused in connection with the telomerase detection aspects of theinvention. For example, in situ hybridization, Southern blotting,Northern blotting and PCR technology. In situ hybridization describesthe techniques wherein the target nucleic acids contacted with the probesequences are located within one or more cells, such as cells within aclinical sample or cells grown in culture. As is well known in the art,the cells may be prepared for hybridization by fixation, e.g. chemicalfixation, and placed in conditions that allow for the hybridization of adetectable probe with nucleic acids located within the fixed cell.

Alternatively, target nucleic acids may be separated from a cell orclinical sample prior to contact with a probe. Any of the wide varietyof methods for isolating target nucleic acids may be employed, such ascesium chloride gradient centrifugation, chromatography (e.g., ion,affinity, magnetic), phenol extraction and the like. Most often, theisolated nucleic acids will be separated, e.g., by size, usingelectrophoretic separation, followed by immobilization onto a solidmatrix, prior to contact with the labelled probe. These prior separationtechniques are frequently employed in the art and are generallyencompassed by the terms “Southern blotting”, that detects DNA and“Northern blotting”, that detects RNA. Virtually of the methods may beadapted for clinical or diagnostic assays, including diagnostic PCRtechnology.

In general, the “detection” of telomerase sequences is accomplished byattaching or incorporating a detectable label into the nucleic acidsegment used as a probe and “contacting” a sample with the labeledprobe. In such processes, an effective amount of a nucleic acid segmentthat comprises a detectable label (a probe), is brought into directjuxtaposition with a composition containing target nucleic acids.Hybridized nucleic acid complexes may then be identified by detectingthe presence of the label, for example, by detecting a radio, enzymatic,fluorescent, or chemiluminescent label.

These detection methods may be employed to detect telomerase-associatedgenes, whether RNA- or protein-encoding, in both clinical and laboratorysamples, e.g., as may be used in telomerase purification, analysis,mutagenesis and the like. In cells or cellular extracts obtained from ananimal or human patient, the detection of telomerase may have particularrelevance, for example, in the diagnosis or detection of tumor cellswithin a sample suspected of containing such cells. This is supported byrecent findings linking telomerase to oncogenesis and various late stagetumors and tumor cells (Harley et al., 1992; Counter et al., 1992,1994a; Shay et al., 1993; Klingelhutz et al., 1994; de Lange, 1994;Greider, 1994). The differential detection and diagnosis of malignanttumors as opposed to benign tumors is also contemplated.

Further clinical samples that may be analyzed for the presence oftelomerase-associated genes, as described above, include those suspectedof containing a pathogen. As telomerase activity is only present individing cells, testing a sample of somatic cells of an animal or humanfor the presence of telomerase may indicate the presence of an invadingunicellular organism within the sample. This may allow disease diagnosisalone, or in combination with other methods. The diagnosis of yeastinfections, for example, is an immediate application of the presentinvention. The development of species-specific markers for otheropportunistic infections is also contemplated.

Diagnostic methods for identifying various conditions associated withinfertility in animals and humans are also provided by the invention.For example, as telomerase activity is required in germ cells, includinghuman sperm and ova, testing samples from animals and humans suspectedof having a condition connected with reproductive failure would provideuseful information. A negative test would likely indicate a defect inthe reproductive capacity of sperm or egg cells within a given sample.

In further embodiments, the invention concerns methods based uponsuppression of “telomeric silencing” for use in identifying non-ciliate,and preferably, yeast telomerase-associated genes or active fragmentsthereof. Such methods generally comprise, initially, preparing a cellcontaining a chromosome that contains a genetic marker located proximalto a telomere, wherein the telomere represses the expression of themarker. Next, one would contact the cell with a composition comprising acandidate gene and identify any gene, or portion thereof, that allowsexpression of the marker. “Genes” identified in this way may be wildtype genes or fragments that may disrupt the telomere function due toover-expression, or they may be mutant or truncated genes that simply donot function correctly.

Appropriate cells for use in such assays include those cells thatcontain an active telomere, such as eukaryotic cells that are capable ofdividing, as exemplified by yeast cells, drosophila cells, and certainhuman cells, such as sperm, egg and cancer cells. The novel technologydeveloped by the inventors is contemplated for use in any organism inwhich the telomeres cause a transcriptional repression (silencing) ofnearby genes. For ease of operation, yeast and Drosophila melanogaster(fruit flies) are currently preferred. However, the use of human cellsis also contemplated.

The genetic markers that are added in the vicinity of a telomere may beany marker gene that gives a readily identifiable phenotype uponexpression. Such markers are also often termed “reporter genes”.Generally, the marker or reporter genes encode a polypeptide nototherwise produced by the cell which is detectable by analysis, e.g., byvisual inspection or by fluorometric, radioisotopic orspectrophotometric analysis. One example is E. coli beta-galactosidase,which produces a color change upon cleavage of an indigogenic substrate;a further example is the enzyme chloramphenical acetyltransferase (CAT),which may be employed with a variety of substrates that give detectableproducts; and still further examples are firefly and bacterialluciferases.

Still further marker genes for use herewith are those capable oftransforming the host cell to express unique cell surface antigens,e.g., viral env proteins such as HIV gp120 or herpes gD, which arereadily detectable by immunoassays. The polypeptide products of thistype of marker gene are secreted, membrane bound polypeptides, orpolypeptides adapted to be membrane targeted, allowing ready detectionby antibodies. However, antigenic reporters are not currently preferredbecause, unlike enzymes, they are not catalytic and thus do not amplifytheir signals.

Yeast markers, when expressed, may result in a colored phenotype orresult in a specific nutrient independence (prototrophy), or even in anutrient requirement, or such like. Exemplary genetic markers that maybe used in yeast include genes that are required for the biosynthesis ofspecific amino acids, such as HIS3, TRP1, LYS2, and LEU2. Genes thatconfer sensitivity to drugs, such as the CAN1 gene that conferssensitivity to canavinine are also contemplated for use. Currentlypreferred marker genes for use in yeast are ADE2 and URA3.

Many suitable genetic markers are also available for use in human cellsystems. These include the markers based upon color detection or antigendetection, as above, and also marker genes that encode polypeptides,generally enzymes, that render the host cells resistant against toxins.These include the neo gene that protects host cells against toxic levelsof the antibiotic G418; the dihydrofolate reductase genes that conferresistance to methotrexate; and the HSV tk gene that is used inconjunction with ganciclovir. Currently preferred examples are themarkers neo and hprt, which are routinely used in the art.

The cells used in such assays may contain two distinct genetic markers,and each genetic marker may be located on a distinct chromosome ifdesired. The combined use of ADE2 and URA3 in yeast cells is currently aparticularly preferred system.

As described hereinabove, human telomerase RNA template andpolypeptide-encoding genes that have substantial sequence homology tothe yeast sequences throughout, or in certain sequence regions, may beisolated by nucleic acid hybridization, i.e., standard cloningtechniques (Sambrook et. al., 1989). However, even if the humansequences are not directly homologous, RNA template and other telomerasegenes may still be isolated using the advantageous methods disclosedherein.

One suitable method for identifying a human telomerase-associated gene,is to apply the suppression of telomeric silencing protocol to a humannucleic acid library using a yeast cell system. Such methods generallycomprise preparing a yeast cell containing a chromosome that contains agenetic marker located proximal to a telomere, where the telomererepresses the expression of the marker; contacting the cell with acomposition comprising a candidate human gene; and identifying a humangene that allows expression of the marker.

Further suitable methods for identifying human telomerase-associatedgenes are those based entirely upon human cells, which methodspresuppose the lowest level of homology between the yeast and human cellsystems. These methods comprise preparing a human cell that contains achromosome having a genetic marker located proximal to a telomere, wherethe telomere represses the expression of the marker; contacting the cellwith a composition comprising a candidate human gene; and identifying ahuman gene that allows expression of the marker.

Another method for isolating genes that encode products that interactwith telomerase RNA is that which assays for genes that re-establishtelomeric silencing when the template RNA is overexpressed, as describedin Example XIV. Here, initially the RNA template is presumed to interactwith a limiting telomerase component to form a non-functional complex.Increasing the concentration of a limiting component, byover-expression, thus re-establishes telomeric silencing. Preferably,RNA template levels that are minimally suppressive are used.

Still more approaches for identifying components that interact withtelomerase RNA are described in Example XIV, which are based uponisolating mutations that enhance or suppress the phenotypes ofconditional telomerase template alleles.

Further elements of this invention are non-ciliate eukaryotic, andpreferably, yeast genes that are identified by any of the foregoingmethods. One such gene is disclosed herein, termed TLC1, that encodes atelomerase RNA template. Other such genes are also disclosed herein,termed STR genes, that encode telomerase-associated polypeptides.Particular examples of such genes of the invention are thus TLC1, STR1,STR3, STR4, STR5 and STR6, and other non-ciliate eukaryotic, andpreferably, yeast nucleic acid segments that have the physical andfunctional characteristics of any of the foregoing genes.

Active fragments of genes and RNA components, such as TLC1 RNA, may alsobe identified using the present methods. Titration assays based uponthose used for the original identification of TLC1 may be used to definethe minimum functional region. It is contemplated that relatively smallregions of the RNA (about 50 bp) that suppress silencing will beidentified. Conditional mutations made in regions of the RNA that areevolutionarily conserved, or that may interact with a limiting factor,as suggested by the titration analysis, will identify functionallyimportant region of the telomerase RNA. Active regions of telomerasegenes and RNA components may also be identified using methods fordissecting small nuclear RNAs (snRNAs), as described in Example XIII.

In still further embodiments, the invention provides methods for use inidentifying candidate substances that bind to yeast and othernon-ciliate eukaryotic telomerase components. These methods generallyinclude preparing an isolated telomerase component; contacting theisolated telomerase component with a composition comprising a candidatesubstance under conditions effective and for a period of time sufficientto allow binding; and detecting the presence of a telomerasecomponent-candidate substance bound complex.

It will be understood that such methods are similar in principle to thenucleic acid hybridization methods described hereinabove. Indeed, the“candidate substances” to be detected may be nucleic acids, includinghuman nucleic acid segments, that are detected by binding to eukaryotic,and preferably, to yeast telomerase RNA or DNA components, andpreferably to a defined small functional region of the template thatsuppress silencing, under the high or low hybridization conditionsdescribed above. However, other components that bind to telomerase maybe identified by binding to the isolated RNA, DNA or polypeptidecomponents of the present invention. These components may includeproteins, polypeptides, peptides, antibodies, small molecules, cofactorsand the like.

Accordingly, the present invention provides binding assays, includinghigh throughput binding assays using recombinant expression products,for use in identifying compounds capable of binding to telomerase or toa telomerase-associated component. The binding assays will preferablyuse the smaller RNA fragments identified in titration or otherfunctional assays described herein.

Further methods for identifying compounds that bind totelomerase-associated components include those based upon cellularassays. One method for identifying a candidate substance that modifiestelomerase activity comprises the following steps:

preparing a eukaryotic, or preferably, a yeast cell containing achromosome that contains a genetic marker located near to, or in thevicinity of, a telomere, the telomere capable of repressing theexpression of the marker;

contacting the cell with a composition comprising a candidate substance;and

identifying a candidate substance that allows expression of the markeror that further represses the expression of the marker.

This method is most suitable for identifying candidate inhibitorysubstances that allow expression of the marker. However, it can also beused to identify candidate stimulatory substances that allow furtherrepression of the marker.

To identify a compound that inhibits telomerase activity, one generallyprepares a cell with a genetic marker that is substantially repressed bythe telomere. Here, the marker gene is located proximal, i.e.,immediately adjacent, to the telomere. Substantial repression is definedby repression to at least about 50%, or preferably, to about 25%, 10% orabout 1%. However, the expression of the marker may be repressed to evenabout 0.01%. The inhibitory substance is then detected by detectinggreater expression of the marker.

To identify a compound that activates telomerase activity, one wouldgenerally prepare a cell with a genetic marker that is either notrepressed at all or that is not substantially or maximally repressed.One would then select a candidate activator by identifying a substancethat establishes or allows repression or more substantial repression.This is based upon the concept that stimulating telomerase to synthesizelonger than normal telomeres will result in an increase in silencing ofa marker gene. To detect the increase requires that a system initiallybe established in which the marker gene is only minimally repressed, oreven not repressed at all. This is readily achieved by inserting themarker gene in the location or vicinity of the telomere, but furtheraway from the telomere rather than immediately adjacent to it. Anincrease in repression, i.e., a decrease in marker gene expression,indicates a positive candidate substance.

Still further methods for identifying compounds that functionallyinteract with telomerase or telomerase-associated components are thosebased upon the telomerase “healing of broken chromosomes” assaydescribed herein. This method is conducted as generally described inExample XII and FIG. 8, using a modified Haber-based assay (Kramer &Haber, 1993). Other useful telomerase functional assays are those thatanalyze telomere length and cell viability with increased age of aculture (Lundblad & Blackburn, 1989), and those in vitro systemsdescribed herein based on the addition of labelled nucleotides to atelomeric-like sequence.

Any of the cellular or activity-based telomerase assays may be adaptedto screen for candidate substances that modify telomerase activity. Toachieve this, one would first conduct the assay in the absence of thetest candidate substance to obtain an activity value in its absence. Onewould then add the candidate substance to the telomerase composition orcell and conduct the assay under the same conditions. Candidatesubstances that reduce or promote telomerase activity can thus bereadily identified.

Useful telomerase-modifying compounds are not believed to be limited inany way to protein or peptidyl compounds or oligonucleotides. In fact,it may prove to be the case that the most useful pharmacologicalcompounds identified through application of a screening assay will benon-peptidyl in nature. Accordingly, in such screening assays, it isproposed that compounds isolated from natural sources, such as animals,bacteria, fungi, plant sources, including leaves and bark, and marinesamples, may be assayed for the presence of potentially usefulpharmaceutical agents. It will be understood that the pharmaceuticalagents to be screened could also be derived from chemical compositionsor man-made compounds.

The invention thus further encompasses components that bind totelomerase and that are capable of modifying telomerase activity, as maybe identified by any of the foregoing binding and/or functional orcellular assay methods. This results in compositions of telomeraseactivators or inhibitors, including pharmaceutically acceptablecompositions, and methods for modifying telomerase activity.

In yet still further embodiments, the present invention thus alsoprovides methods for modifying the replicative capacity of a cell, whichmethods comprise contacting a telomerase-containing cell with an amountof a component or substance effective to modify telomerase activity.“Modifying” in this context includes both compositions and methods forinhibiting telomerase activity, as may be used, e.g., in inhibiting orkilling a tumor cell or a pathogen; and compositions and methods forstimulating telomerase activity, as may be used in embodiments connectedwith promoting the replication of a cell, such as in treatinginfertility.

Where the telomerase-containing cells are located within an animal, apharmaceutically acceptable composition of the telomerase activator orinhibitor may be administered to the animal in an amount effective tomodify the telomerase activity of the target cell. In terms ofinhibiting telomerase activity in tumor cells, this is contemplated tobe an effective mechanism by which to treat cancer that will have verylimited side effects. Similarly, effective antimicrobial treatments arecontemplated, as are applications in treating age-related disorders suchas atherosclerosis and osteoporosis. Further, gene therapy usingfunctional telomerase-associated genes is envisioned to be of use intreating telomerase dysfunction, as could provide a treatment forinfertility in humans and other animals.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A, FIG. 1B and FIG. 1C. Overexpression of TLC1 suppressestranscriptional silencing at telomeres, but not at the HML locus. InFIG. 1A, Viability on medium lacking uracil was measured for S.cerevisiae strains containing URA3 either at telomere VII-L (UCC3505) orat HML (UCC3515), and overexpressing either vector alone (pTRP, downsloping hatched bar), a representative TLC1 cDNA clone (pTRP6, upwardsloping hatched bar), or a SIR4 cDNA clone (pTRP10, white bar) (Kyrionet al., 1993). In FIG. 1B, ADE2 expression, as assayed by colony color,was examined in cells containing ADE2 placed near telomere V-R(UCC3505), and containing the vector (pTRP). In FIG. 1C, ADE2expression, as assayed by colony color, was examined in cells containingADE2 placed near telomere V-R (UCC3505), and containing anotherrepresentative TLC1 cDNA clone (pTRP61). The medium contained 3%galactose and lacked tryptophan. The median value for viability in theabsence of uracil is marked by the height of each column, and the upperextreme is indicated by the error bar. The strains were pregrown forfour days on solid synthetic medium without tryptophan (to maintainselection for the plasmid) that contained 3% galactose (to induce theGAL1 promoter controlling expression of the cDNA inserts). Colonies werethen diluted in water, and serial dilutions were plated on 3% galactosemedium without tryptophan and uracil. Cells were also plated on mediumcontaining uracil, to determine overall cell viability. Five independenttransformants of each strain were tested.

FIG. 2. Overexpression of TLC1 causes a decrease in telomeric tractlength. Yeast strain UCC3505 carrying either vector (pTRP, lanes 1 and2) or a TLC1 cDNA clone (pTRP6, lanes 3 and 4) were pregrown forapproximately 60 generations on medium containing 3% galactose withouttryptophan. Genomic DNA was prepared from two independent transformantsof each strain, digested with Apa I and Xho I, separated byelectrophoresis on a 1% agarose gel, and blotted onto a nylon membrane.The membrane was probed with a 1.1 kb Hind III-Sma I URA3 fragment. TheURA3 gene in this strain is located adjacent to telomere VII-L. Thehigher molecular weight (non-telomeric) URA3 fragments representsequences of the telomeric URA3 that are centromere-proximal to the URA3Apa I site, and sequences from the ura3-52 allele at the normalchromosomal locus of URA3. The Southern blot was also probed with an 81bp labeled (TG)₁₋₃TG₂₋₃(telomeric sequence) riboprobe, to determine thetelomere length of chromosomes with Y′ elements (Walmsey et al., 1984).These telomere-associated sequences are at the ends of multiple yeastchromosomes and generally have Xho I sites at their telomere-proximalends (Louis & Haber, 1990). Y′-containing chromosomes showed a decreaseof telomere length upon overexpression of the TLC1 cDNA clone similar tothat seen for telomere VII-L.

FIG. 3A and FIG. 3B. TLC1 encodes a 1.3 kb RNA. TLC1 transcript levelswere analyzed in yeast strains containing a wild-type TLC1 gene (lane1), or a tlc1::LEU2 disruption allele (lane 2), and in wild-type cellscarrying either vector (pTRP, lane 3) or a TLC1 cDNA clone (pTRP61, lane4). Total RNA was isolated from mid-log phase cells grown in rich medium(for strains lacking plasmids) or synthetic medium without tryptophanbut with 3% galactose (for the plasmid-containing strains). 20 μg of RNAfrom each strain was electrophoretically separated on a 0.9% agaroseformaldehyde gel and transferred to a nylon membrane. FIG. 3A shows themembrane probed with a 1.25 kb TLC1 antisense probe (made from thepTRP61 insert) and exposed to film. Phosphor-imaging analysis determinedthat there is approximately 12-fold more TLC1 RNA in the overexpressingstrain (lane 4) than in the vector-containing wild-type strain grownunder the same conditions (lane 3). FIG. 3B displays the ethidiumbromide-stained gel prior to blotting, with the sizes of the rRNAspecies (25S and 18S) indicated on the right. The wild-type and tlc1strains shown in lanes 1 and 2 were derived from sporulation of UCC3508(Aparicio et al., 1991). The yeast strain transformed with the pTRP andpTRP61 plasmids, shown in lanes 3 and 4, is UCC3505.

FIG. 4A. Disruption of TLC1 causes progressive telomere shortening and agradual decrease in growth rate and viability. A TLC1/tlc1::LEU2 diploid(UCC3508) was sporulated and the resulting tetrads dissected andgerminated on rich medium. Colonies representing the four spore productsfrom a tetrad were inoculated into 5.5 ml of rich medium and grown at30° C. Every 24 hours, 5 ml of the culture were used for the preparationof genomic DNA, and 5 μl were used to inoculate 5.5 ml of fresh medium.The genomic DNA was digested with Apa I, electrophoresed on a 1% agarosegel, transferred to a nylon membrane and hybridized to a 1.1 kb URA3probe. The URA3 gene is located adjacent to telomere VII-L in thesestrains. In a similar study, genomic DNA from TLC1 and tlc1 cultures wasdigested with Xho I, as well as Apa I, in order to examine Y′-containingtelomeres using the Southern blotting method described in FIG. 2 withthe 81 bp labeled telomeric sequence riboprobe (Walmsey et al., 1984).The size of this population of telomeres decreased in size at the samerate as the URA3-labeled telomere VII-L.

FIG. 4B. Disruption of TLC1 causes progressive telomere shortening and agradual decrease in growth rate and viability. In a study similar tothat of FIG. 4A, UCC3508 spore products were grown continuously in richmedium. Every 24 hours the cell density was determined and each culturewas diluted to 3×10⁵ cells/ml in 5.5 ml of fresh medium for furthergrowth. The cell density at each time point is plotted for the two TLC1(white circle and square) and tlc1 (hatched ◯ and □) spore products of atetrad.

FIG. 5A and FIG. 5B. The TLC1 gene encodes an RNA that functions as atemplating component of telomerase, an enzyme that elongates the G-richstrand of S. cerevisiae telomeres. In FIG. 5A, is shown a model by whichthe TLC1 RNA anneals to the single-stranded G-rich overhanging strand atthe end of the chromosome and templates its elongation via a reversetranscription reaction. The bold-type DNA bases represent newlysynthesized sequence. FIG. 5B, shows that, accordingly, mutating theputative template motif of TLC1, creating the TLC1-1(HaeIII) allele,results in the incorporation of the altered sequence into telomeric DNA.

FIG. 6A. Altering the putative telomere-templating region of TLC1results in the incorporation of the mutant sequence into telomerictracts. Fragment-mediated transformation of TLC1/TLC1 andTLC1-1(HaeIII)/TLC1 diploid strains was used to replace the terminalsequences of the left arm of one of the chromosome VII homologs with aURA3 gene and a short telomeric tract sequence. The mosttelomere-proximal Apa I and Hae III sites in the fragment used in thetransformation overlap and are located 0.75 kb from the telomeric end ofthe fragment.

FIG. 6B. Altering the putative telomere-templating region of TLC1results in the incorporation of the mutant sequence into telomerictracts. Restriction digests of genomic DNA from transformed strains wereused to determine whether Hae III sites were introduced into the newtelomere VII-L upon its elongation in vivo. Genomic DNA from TLC1/TLC1and TLC1-1(HaeIII)/TLC1 yeast strains, either transformed with URA3TEL(Telomeric URA3+) or not (Telomeric URA3−), was digested with Apa I (A)or Hae III (H). The DNA fragments were separated by electrophoresis on a1.25% agarose gel, transferred to a nylon membrane, and probed with alabeled 0.6 kb URA3 probe (Apa I-Hind III fragment), as depicted in FIG.6A. Each Telomeric URA3+ strain represents an independently isolatedtransformant.

FIG. 7A. Quantitative suppression of telomeric silencing by variousdifferent genes. This was assessed by viability in the absence of uracilfor the strains that contained the telomeric URA3 gene and each of the10 highly expressed genes of Example X. All the genes suppressedsilencing of the telomeric URA3, although a hierarchy of suppression wasobserved.

FIG. 7B. Effect of genes on silencing at HML. The expression plasmidscontaining each of the 10 genes of Example X were introduced into astrain in which the URA3 gene was inserted into the HML locus (Mahoney &Broach, 1989). Overexpression of TLC1 (STR2) had no effect on silencingat HML, but strongly suppressed telomeric silencing of URA3 and ADE2.The SIR4 and ASF1 genes derepressed HML very well, as did the STR1,STR4, and RRP3 genes. Overexpression of RPL32, STR3, STR5 and STR6 hadintermediate effects at HML.

FIG. 8. Schematic representation of the new genetic system to testtelomere healing, as described in Section 2 of Example XII.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Telomeres, the natural ends of linear eukaryotic chromosomes, areessential for chromosome stability. Because of the nature of DNAreplication, telomeres require a specialized mechanism to ensure theircomplete duplication. This is controlled by telomerase activity. Due toits role in controlling replication, changes in telomerase activity havebeen linked to disturbances in cell proliferation, as can lead to acancerous phenotype (de Lange, 1994; Greider, 1994; Harley et al.,1992).

The evolutionary conservation of telomere structure suggested to thepresent inventors that the study of telomerase in genetically tractableorganisms, such as the budding yeast Saccharomyces cerevisiae, wouldyield important information directly applicable to telomere studies ineukaryotic and mammalian cells. The existence of an S. cerevisiaetelomerase was suggested by studies in which double-strand breaks wereintroduced into yeast chromosomes in vivo, after which healedchromosomes with new telomeric tracts were formed (Kramer & Haber,1993). Specific 13-bp motifs (GTGTGTGGGTGTG; SEQ ID NO:2), or subsetsthereof, were found at the junction between the break site and the newtelomeric tracts, suggesting that this sequence is added de novo (Kramer& Haber, 1993).

However, prior to the present invention, little was known about themolecular machinery that could be involved in telomeric replication inS. cerevisiae. Previously, the only candidate as a component of thetelomere replication apparatus was the protein encoded by the EST1 gene(Lundblad & Szostak, 1989). Its role in telomere replication wassuggested by the finding that est1 cells display progressive telomereshortening, accompanied by a gradual loss of chromosome stability andcell viability. The direct function of Est1p still remains to beelucidated.

The inventors discovered that S. cerevisiae telomeres repress, orsilence, expression of genes located nearby (Example I). Silencing oftelomeric genes is due to a structurally distinct chromatin domain whoseformation initiates at the telomere (Example III). Evidence for thisspecialized chromatin structure includes: identification of mutations inthe histone H3 and H4 genes which relieve telomeric silencing (ExampleII), the finding that telomere-adjacent chromatin contains histone H4 ina hypoacetylated state compared to H4 in actively transcribed chromatin(Braunstein et al., 1993), and the relative inaccessibility oftelomere-proximal DNA to in vivo modification by the E. coli dammethyltransferase protein (Gottschling, 1992). At least six additionalgene products, including the telomere DNA binding protein, RAP1, arerequired for telomeric silencing (Aparicio et al., 1991; Kyrion et al.,1993).

In order to identify genes in S. cerevisiae that are important fortelomere function, the inventors developed and used a novel screeningmethod to identify genes that, when expressed at high levels, suppresstelomeric silencing. This screen lead to the identification of the geneTLC1 (telomerase component 1), one of the components of the presentinvention, along with several other novel genes.

TLC1 encodes the template RNA of telomerase, a ribonucleoproteinrequired for telomere replication in a variety of organisms. Thediscovery of TLC1 is the first clear evidence that shows telomeraseexists in S. cerevisiae. This finding will facilitate further telomerasestudies and screening assays to identify activators or inhibitors withpotential for modulating telomerase activity, as may ultimately be usedin a clinical setting.

The present discoveries may be utilized in conjunction with certaintechniques that are well-known in the biological arts and that arefurther described in the following sections.

A. Biological Functional Equivalents

Modification and changes may be made in the structure oftelomerase-associated polypeptides and still obtain molecules havinglike or otherwise desirable characteristics. For example, certain aminoacids may be substituted for other amino acids in a protein structurewithout appreciable loss of interactive binding capacity with structuressuch as, for example, antigen-binding regions of antibodies or bindingsites on substrate molecules, receptors, RNA molecules, chromosomal endsand the like. Since it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence (or, of course, its underlying DNA coding sequence) andnevertheless obtain a protein with like (agonistic) properties. Equally,the same considerations may be employed to create a protein orpolypeptide with countervailing (e.g., antagonistic) properties. It isthus contemplated by the inventors that various changes may be made inthe sequences of the telomerase-associated proteins or peptidesdisclosed herein (or their underlying DNA) without appreciable loss oftheir biological utility or activity.

It is also well understood by the skilled artisan that, inherent in thedefinition of a biologically functional equivalent protein or peptide,is the concept that there is a limit to the number of changes that maybe made within a defined portion of the molecule and still result in amolecule with an acceptable level of equivalent biological activity.Biologically functional equivalent peptides are thus defined herein asthose peptides in which certain, not most or all, of the amino acids maybe substituted. In particular, where smaller peptides are concerned, itis contemplated that relatively few amino acids may be changed within agiven peptide. Of course, a plurality of distinct proteins/peptides withdifferent substitutions may easily be made and used in accordance withthe invention.

It is also well understood that where certain residues are shown to beparticularly important to the biological or structural properties of aprotein or peptide, e.g., residues in active sites or key bindingregions, such residues may not generally be exchanged.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and the like. An analysisof the size, shape and type of the amino acid side-chain substituentsreveals that arginine, lysine and histidine are all positively chargedresidues; that alanine, glycine and serine are all a similar size; andthat phenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and charge characteristics,these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index or score and still retain asimilar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. Thus, it is understood that an amino acid can be substitutedfor another having a similar hydrophilicity value and still obtain abiologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid. A table of amino acids andtheir codons is presented herein (Table 1) for use in such embodiments,as well as for other uses, such as in the design of probes and primersand the like.

TABLE 1 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In addition to the peptidyl compounds described herein, the inventorsalso contemplate that other sterically similar compounds may beformulated to mimic the key portions of the peptide structure. Suchcompounds, which may be termed peptidomimetics, may be used in the samemanner as the peptides of the invention and hence are also functionalequivalents. The generation of a structural functional equivalent may beachieved by the techniques of modelling and chemical design known tothose of skill in the art. It will be understood that all suchsterically similar constructs fall within the scope of the presentinvention.

U.S. Pat. No. 4,554,101 (Hopp, incorporated herein by reference) alsoteaches the identification and preparation of epitopes from primaryamino acid sequences on the basis of hydrophilicity. Through the methodsdisclosed in Hopp one of skill in the art would be able to identifyepitopes from within the telomerase-associated amino acid sequencesdisclosed herein. Such regions would also be referred to as “epitopiccore regions”.

Numerous scientific publications have been devoted to the prediction ofsecondary structure, and to the identification of epitopes, fromanalyses of amino acid sequences (Chou & Fasman, 1974a,b; 1978a,b,1979). Any of these may be used, if desired, to supplement the teachingsof Hopp in U.S. Pat. No. 4,554,101. Moreover, computer programs arecurrently available to assist with predicting antigenic portions andeptiopic core regions of proteins. Examples include those programs basedupon the Jameson-Wolf analysis (Jameson & Wolf, 1998; Wolf et al.,1988), the program PepPlot® (Brutlag et al., 1990; Weinberger et al.,1985), and other new programs for protein tertiary structure prediction(Fetrow & Bryant, 1993). The identification of epitopic regions fromwithin the telomerase-associated sequences allows the ready generationof specific antibodies.

B. Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art. As will be appreciated, the technique typically employs a phagevector which exists in both a single stranded and double stranded form.Typical vectors useful in site-directed mutagenesis include vectors suchas the M13 phage. These phage are readily commercially available andtheir use is generally well known to those skilled in the art. Doublestranded plasmids are also routinely employed in site directedmutagenesis which eliminates the step of transferring the gene ofinterest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartthe two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes a telomerase-associated component.An oligonucleotide primer bearing the desired mutated sequence isprepared, this primer is then annealed with the single-stranded vector,and subjected to DNA polymerizing enzymes such as E. coli polymerase IKlenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation. This heteroduplex vector is then used totransform appropriate cells, such as E. coli cells, and clones areselected which include recombinant vectors bearing the mutated sequencearrangement.

The preparation of sequence variants of the selectedtelomerase-associated gene using site-directed mutagenesis is providedas a means of producing potentially useful species and is not meant tobe limiting as there are other ways in which sequence variants may beobtained. For example, recombinant vectors encoding a desiredtelomerase-associated gene may be treated with mutagenic agents toobtain sequence variants, as used in the mutagenesis of plasmid DNAusing hydroxylamine.

C. Nucleic Acid Hybridization

In Southern analysis, membrane-bound, denatured DNA fragments arehybridized to a labeled DNA probe. Following this hybridization, themembrane is washed in order to remove nonspecifically bound probe,leaving only probe that is specifically base-paired to the target DNA.By controlling the stringency of the washing conditions, differentlevels of probe-target DNA complementarity may be detected.

High stringency conditions are useful in order to identify DNA fragmentswith little mismatch, even close to and including 100% complementarityto the probe DNA. Low stringency conditions are used to identifysequences that are related, though not identical, to the probe DNA,e.g., members of a multigene family, or a single gene in a differentorganism.

Preferred hybridization conditions are, currently, those that use abuffer of 5×SSC, 0.5%(w/v) blocking reagent, 0.1%(w/v)N-lauroylsarcosine, Na-salt, 0.02% (w/v) SDS and 50%(w/v) formamide,with hybridization at 42° C. overnight. The high stringency washingconditions involve washing the blot twice for 5 minutes with Blot Wash#1 (2×SSC, 0.1%(w/v) SDS), and then washing twice for 15 minutes withBlot Wash #2 (0.1×SSC, 0.1%(w/v) SDS) at 55° C.

For low stringency hybridization, the hybridization conditions remainusing 5×SSC, 0.5%(w/v) blocking reagent, 0.1%(w/v) N-lauroylsarcosine,Na-salt, 0.02%(w/v) SDS and 50% (w/v) formamide, with hybridization at42° C. overnight. The low stringency washing conditions involve usingBlot wash #2 as 0.2×SSC, 0.1%(w/v) SDS at 45° C. In the low stringencyprotocols, a certain limited variation in the conditions may benecessary to achieve optimal conditions, on a case-by-case basis. Suchoptimization is standard and routinely practiced by those of skill inthe art.

D. Protein Expression

To express a recombinant telomerase-associated RNA or protein componentin accordance with the present invention one would prepare an expressionvector that comprises the telomerase-associated component under thecontrol of one or more promoters. The “upstream” promoters stimulatetranscription of the DNA and promote expression of the encodedrecombinant protein or RNA unit. This is the meaning of “recombinantexpression” in this context.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieve RNAor protein expression in a variety of host-expression systems. Celltypes available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli×1776 (ATCC No. 31537) as well as E. coli W3110(F-, lambda-, prototrophic, ATCC No. 273325); bacilli such as Bacillussubtilis; and other enterobacteriaceae such as Salmonella typhimurium,Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using pBR322, a plasmid derived from an E. coli species.pBR322 contains genes for ampicillin and tetracycline resistance andthus provides easy means for identifying transformed cells. The pBRplasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the microbialorganism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making a recombinant phagevector which can be used to transform host cells, such as E. coli LE392.Further useful vectors include pIN vectors; and pGEX vectors, for use ingenerating glutathione S-transferase (GST) soluble fusion proteins forlater purification and separation or cleavage.

Those promoters most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enabling askilled worker to ligate them functionally with plasmid vectors.

Naturally, in certain embodiments, yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containingtelomerase-associated RNA or protein coding sequences will be preferredin certain embodiments.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used (Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemperet al., 1980). This plasmid already contains the trpl gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, 1977). The presence of the trpl lesion as a characteristic ofthe yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In yeast, any plasmid vector containing a yeast-compatible promoter, anorigin of replication, and termination sequences is suitable. However,preferred recombinant expression vectors include pYPGE-2, as describedby Brunelli & Pall (1993).

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing the telomerase-associated codingsequences.

In a useful insect system, Autograph californica nuclear polyhidrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The telomerase-associated proteinor RNA coding sequences are cloned into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of the coding sequences results in the inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed (e.g., U.S. Patent No.4,215,051 (Smith)).

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2,3T3, RIN and MDCK cell lines. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and processes the gene product in the specific fashion desired.Such modifications (e.g., glycosylation) and processing (e.g., cleavage)of protein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecells lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

Expression vectors for use in such cells ordinarily include an origin ofreplication (as necessary), a promoter located in front of the gene tobe expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). Further, itis also possible, and often desirable, to utilize promoter or controlsequences normally associated with the desired gene sequence, providedsuch control sequences are compatible with the host cell systems.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2, and mostfrequently Simian Virus 40 (SV40). The early and late promoters of SV40virus are particularly useful because both are obtained easily from thevirus as a fragment which also contains the SV40 viral origin ofreplication. Smaller or larger SV40 fragments may also be used, providedthere is included the approximately 250 bp sequence extending from theHind III site toward the Bg1 I site located in the viral origin ofreplication.

In cases where an adenovirus is used as an expression vector, the codingsequences may be ligated to an adenovirus transcription/translationcontrol complex, e.g., the late promoter and tripartite leader sequence.This chimeric gene may then be inserted in the adenovirus genome by invitro or in vivo recombination. Insertion in a non-essential region ofthe viral genome (e.g., region E1 or E3) will result in a recombinantvirus that is viable and capable of expressing telomerase-associated RNAor proteins in infected hosts.

Specific initiation signals may also be required for efficienttranslation of telomerase-associated component coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Exogenous translational control signals, including the ATG initiationcodon, may additionally need to be provided. One of ordinary skill inthe art would readily be capable of determining this and providing thenecessary signals. It is well known that the initiation codon must be inphase (or in-frame) with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably expressconstructs encoding telomerase-associated components may be engineered.Rather than using expression vectors that contain viral origins ofreplication, host cells can be transformed with vectors controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then are switched to a selective media. The selectable markerin the recombinant plasmid confers resistance to the selection andallows cells to stably integrate the plasmid into their chromosomes andgrow to form foci which in turn can be cloned and expanded into celllines.

A number of selection systems may be used, including, but not limited,to the herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosophoribosyltransferase, intk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistancecan be used as the basis of selection for dhfr, that confers resistanceto methotrexate; gpt, that confers resistance to mycophenolic acid, neo,that confers resistance to the aminoglycoside G-418; and hygro, thatconfers resistance to hygromycin.

E. Monoclonal Antibody Generation

Means for preparing and characterizing antibodies are well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster injection, may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate MAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified telomerase-associated protein, polypeptide orpeptide. The immunizing composition is administered in a mannereffective to stimulate antibody producing cells. Rodents such as miceand rats are preferred animals, however, the use of rabbit, sheep frogcells is also possible. The use of rats may provide certain advantages(Goding, 1986, pp. 60-61), but mice are preferred, with the BALB/c mousebeing most preferred as this is most routinely used and generally givesa higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11-X45-GTG 1.7 and S194/5XXO Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp.71-74, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

A molecular cloning approach may also be used to generate monoclonals.For this, combinatorial immunoglobulin phagemid libraries are preparedfrom RNA isolated from the spleen of the immunized animal, and phagemidsexpressing appropriate antibodies are selected by panning using cellsexpressing the antigen and control cells. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by H and L chaincombination which further increases the chance of finding appropriateantibodies.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments that are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I Position Effect at S. Cerevisiae Telomeres

Position effect is a term used to describe phenomena in which a gene'sbehavior is affected by its location on the chromosome (Lima-de-Faria,1983b). The change in behavior can be manifested in a variety of ways,such as a difference in phenotype, transcription level, recombinationfrequency, or replication timing. Although position effects have beenreported in insects, plants, and mice, most studies have been carriedout in Drosophila, where euchromatic genes translocated near or withincentromeric heterochromatin come under a position effect, typicallyexhibiting phenotypic repression (Spofford, 1976). More recently, in S.cerevisiae the silent mating type loci, HML and HMR, have been shown toexert a position effect on the transcription of nearby genes (Brand etal., 1985; Mahoney and Broach, 1989; Schnell and Rine, 1986).

Telomeric DNA in ciliates, humans, and probably other eukaryotes,facilitates the complete replication of linear DNA molecules by servingas substrates for telomerase (Zakian, 1989; Blackburn, 1990). Telomeresact as chromosome “caps”; in contrast to ends generated by chromosomebreakage, telomeres are protected from exonucleolytic degradation andend-to-end fusions. Telomeres are also implicated in establishingnuclear organization by engaging in associations with other telomeresand with the nuclear envelope (Agard and Sedat, 1983; Lima-de-Faria,1983a).

In S. cerevisiae, the simple DNA repeat (TG₁₋₃) is found at the ends ofall linear chromosomes (Shampay et al., 1984; Walmsley et al., 1984).The repeated sequence is necessary and sufficient in cis to providetelomere function in vivo (Wellinger and Zakian, 1989): telomericrepeats are required at each end of a DNA molecule in order for it to bemaintained in a linear form in yeast (Lundblad and Szostak, 1989; Plutaand Zakian, 1989). Examination of chromosomal ends reveals aheterogeneity in the number of (TG₁₋₃) repeats at individual telomeresboth within and between strains, with an average of ˜300 bp (Shampay andBlackburn, 1988; Walmsley and Petes, 1985). In addition to the (TG₁₋₃)repeats at the ends of chromosomes, most yeast telomeres bear middlerepetitive elements called telomere associated sequences (Chan and Tye,1983a; Chan and Tye, 1983b).

In S. cerevisiae there are two types of telomere associated sequences:Y′ is a highly conserved sequence that exists in a long (˜6.7 kbp) andshort form (5.2 kbp), whereas X is less well conserved and ranges insize from 0.3 to 3.8 kbp. The sequences can occur in tandem arrays nearthe ends of the chromosome, where they are separated from one another bytracts of (TG₁₋₃) 50-130 bp in length (Walmsley et al., 1984). It isunclear whether the X and Y′ sequences serve a particular function,since they are absent from some telomeres (Jager and Philippsen, 1989;Zakian and Blanton, 1988); however in humans and Drosophila telomereassociated sequences have been implicated in meiotic chromosome pairingand the establishment of heterochromatin (Ellis et al., 1989; Young etal., 1983).

In order to understand better the properties of telomeres, the inventorsbegan an investigation to map in vivo protein-DNA interactions atchromosomal termini in S. cerevisiae. The inventors chose to examine asingle telomere by introducing a unique marker adjacent to the tract of(TG₁₋₃) DNA at the end of a chromosome. However, early in the course ofsuch investigations it was realized that the transcription of the geneused to mark the telomere was altered. In this example, the inventorsdemonstrate that in S. cerevisiae, telomeres without an X or Y′ exert aposition effect on the expression of genes located nearby.

When URA3, TRP1, HIS3, or ADE2 was located near a telomere, the gene'stranscription was repressed. However, the expression of each gene wasreversible between states of repressed and active transcription. Bothtranscriptional states were inherited mitotically in a semi-stablemanner. Switching between the states appears to be under epigeneticcontrol. At a locus ˜20 kbp from the telomere, transcription of URA3 wasnot repressed, even when an 81 bp tract of (TG₁₋₃) sequence was locatedadjacent to the gene. However, the internal 81 bp tract spontaneouslybecame a telomere at a frequency of ˜10⁻⁶, and in so doing repressed theexpression of the URA3 gene. This example therefore provides geneticmethods for analyzing telomere structure, and formation of new telomeresfrom internal telomeric DNA sequences.

A. Material & Methods

1. Construction of Plasmids

Plasmid pVII-L URA3-TEL was constructed in two steps, beginning with theplasmid pYTCA-1. Plasmid pYTCA-1 has the 125 bp Hae III-Mnl I fragmentfrom pYt103, that contains 81 bp of (TG₁₋₃) sequence derived from ayeast telomere, in the Sma I site of pUC9 (Runge and Zakian, 1989;Shampay et al., 1984). The (TG₁₋₃) sequence is oriented such thatdigestion of pYTCA-1 with Eco RI will yield an end that is a substratefor telomere formation in yeast. Plasmid pYTCA-1 was digested with HindIII and Hinc II and a 1.1 kbp Hind III-Sma I DNA fragment that containsthe URA3 gene was ligated between these sites (Rose et al., 1984) toform pURA3-TEL. Plasmid YA4-2 (obtained from V. Williamson) contains theADH4 gene on an Eco RI-Bgl II fragment inserted within the Eco RI-Bam HIsites of pUC8 (Walton et al., 1986; Williamson and Paquin, 1987).Plasmid pURA3-TEL was digested with Hind III and the 1.2 kbp Hind IIIfragment of pYA4-2 was ligated within, such that the Sal I site of theinserted fragment was positioned away from the URA3 gene. This resultsin plasmid pVII-L URA3-TEL.

Plasmid adh4::URA3-TEL was also constructed in two steps. First, pVII-LURA3-TEL was digested with Bam HI, the DNA ends were made blunt bytreatment with T4 DNA polymerase and dNTPs, and the plasmid wasrecircularized. Next, this new plasmid, pVII-L URA3-TEL (-Bam HI), wasdigested with Eco RI, the ends were made blunt as before, and ligated tothe blunt-ended 1.8 kbp Hind III-Eco RI fragment of YA4-2. Plasmids withthe Bam HI site furthest from the (TG₁₋₃) sequence have the correctorientation of the insert.

Plasmid adh4::URA3 was constructed by digesting pVII-L URA3-TEL with BamHI, making the ends blunt, then treating the plasmid with Eco RI; the1.8 kbp Hind III-Eco RI fragment of YA4-2 with only its Hind III endmade blunt, was ligated into the plasmid.

Plasmid V-R URA3-TEL was made by digesting pVII-L URA3-TEL with Hind IIIand replacing the ADH4 derived sequence with the 2.8 kbp Hind IIIfragment of plasmid B6-10H, such that the Eco RI site of the insert wasfurthest from the URA3 gene. Plasmid B6-10H (obtained from C. Newlon)contains ˜19 kbp of unique DNA sequence from the region adjacent to thesubtelomeric Y′ repeat on the right arm of chromosome V (Chan and Tye,1983b; McCarroll and Fangman, 1988). The 2.8 kbp Hind III fragment fromB6-10H used in this study is unique sequence ˜5.5 kbp from the Y′repeat.

Plasmid pULA was constructed in a two step process. First, the 1.1 kbpHind III-URA3 fragment was inserted into the Hind III sit of a pUC9derivative, in which the Pst I site has been deleted. The resultingplasmid was digested with Pst I and Nsi I; the coding sequence of URA3was removed and replaced with a 4 kbp Pst I fragment containing LEU2isolated from YEP13 (Broach et al., 1979).

Plasmids pADHIS3(+), pADHIS3(−), pADADE2(+), pADADE2(−), pADTRP1(+), andpADTRP1(−) were all constructed by inserting the wild-type HIS3, ADE2,or TRP1 genes in either orientation, into the Bam HI site in the vectorVII-L URA3-TEL. For HIS3: A 1.85 kbp Bam HI fragment from plasmid pHIS3(Struhl, 1985; obtained from K. Runge) was inserted into the Bam HI siteof VII-L-URA3-TEL. Two plasmids are formed: pADHIS3(+), in which theHIS3 gene is in the same transcriptional orientation as the URA3 gene,and pADHIS3(−) which has the HIS3 gene in the opposite orientation.

For ADE2, a 3.6 kbp Bam HI fragment in plasmid pL909 (obtained from R.Keil), was inserted into the Bam HI site of the vector VII-L URA3-TEL.The resulting plasmids were designated pADADE2(+), indicating ADE2transcription in the same direction as the adjacent URA3 gene, orpADADE2(−) for ADE2 transcription in the opposite direction.

For TRP1, 0.85 kbp Eco RI-Bgl II fragment from plasmid YRp7 (Struhl etal., 1979) was blunt-ended with T4 DNA polymerase and inserted into theBam HI site in VII-L URA3-TEL which also had the Bam HI ends filled-inwith T4 DNA polymerase. The plasmid with the TRP1 gene in the sametranscriptional orientation as URA3 was denoted pADTRP1(+), while theplasmid in which TRP1 transcription was in the opposite direction asURA3 transcription was pADTRP1(−).

Plasmid TRP1/RS306 was made by inserting the Eco RI-Bgl II fragment ofTRP1 into the Eco RI-Bam HI site of pRS306 (Sikorski and Hieter, 1989).

E. coli strain MC1066 (r⁻ m⁻, trpC9830, leub600, pyrF::Tn5, lacΔX74,strA, galU, galk) was used as a host for all plasmids (Casadaban et al.,1983). LB medium with ampicillin (100 μg/ml) and M9 medium supplementedwith appropriate amino acids were prepared as described by Maniatis etal. (Maniatis et al., 1982). Complementation of MC1066 mutations by thehomologous yeast genes was used when applicable.

2. Yeast Strains & Methods

Media used for the growth of S. cerevisiae were based on syntheticcomplete media as described by Sherman et al. (Sherman et al., 1986) towhich uracil (35 mg/l), tyrosine and lysine (60 mg/l), and leucine andisoleucine (80 mg/l) had been added. One gram of 5-FOA per liter ofmedia was added to determine resistance to 5-FOA. Medium for ADE2red/white sectored colony growth was as described (Klapholz andEsposito, 1982) except arginine was 50 mg/l and threonine was 100 mg/l.Colonies were grown for three days at 300, then incubated for 1-2 weeksat 4° for full color development. S. cerevisiae transformation wasperformed using the lithium acetate procedure (Ito et al., 1983).

To delete the URA3 gene, strain 1GA2 (MATα ade2 ade5 leu2-3,112 lys5cyh2^(r) can1^(r); made in this study) was transformed with Hind IIIdigested pULA (see above), and Leu+ colonies were isolated. Thestructure of the chromosome from which URA3 was deleted was checked bySouthern analysis in Leu+ isolates that also tested Ura⁻. DG20 is theura3Δ::LEU2 derivative of 1GA2. Strains DG26, DG27, DG28, and DG30 wereconstructed by transforming DG20 with different DNA fragments: DG26 withplasmid adh4::URA3-TEL cleaved by Bam HI and Sal I, DG27 with plasmidadh4::URA3 cleaved by Bam HI and Sal I, DG28 with plasmid VII-L URA3-TELcleaved by Sal I and Eco RI, and DG30 with plasmid V-R URA3-TEL cleavedby Eco RI. All transformants were selected as being both Ura⁺ and Leu⁺.The expected structure for each transformant was verified by Southernanalysis. In each case, total genomic DNA was cleaved twice, once by BglII and once by Pst I. The Southern blots of DG26, DG27, and DG28 werehybridized with a series of DNA probes which included: the 1.1 kbp HindIII URA3 gene, the proximal ADH4 probe, and the distal ADH4 probe. Thestructure of DG30 was verified in a similar manner using probes fromplasmid B6-10H.

Strains UCC41, UCC42, UCC45, UCC61, UCC62, UCC63, UCC81, UCC82, andUCC83 were derived from strain 4-1 (MATα lys2 his4 trp1Δ ade2 leu2-3,112ura3-52 made in this study), by transforming strain 4-1 with differentDNA fragments and selecting for Ura⁺ colonies: UCC41 with pADADE2(+) cutwith Sal I and Not I, UCC42 with pADADE2(−) cut with Sal I and Not I,UCC45 with pL909 cut with Bam HI, UCC61 by pADTRP1(+) cut with Sal I andEco RI, UCC62 with pADTRP1(−) cut with Sal I and Eco RI, UCC81 withVII-L URA3-TEL cut with Sal I and Eco RI, UCC82 with adh4::URA3 cut withBam HI and Sal I, UCC83 with pUCU (contains the 1.1 kbp Hind IIIfragment containing the URA3 gene in pUC9) cut with Hind III, and UCC63with pTRP1/RS306 digested with Nde I.

Strains UCC51, UCC52, UCC53, UCC74, UCC75, and UCC76 were derived fromstrain 3482-16-2 (MATa, met2, his3D-1, leu2-3,112, trp1-289, ura3-52,obtained from L. Hartwell), by transforming strain 3482-16-2 withdifferent DNA fragments, again selecting for Ura⁺ colonies: UCC51 bypADHIS3(+) cut with Sal I and Eco RI, UCC52 with pADHIS3(−) cut with SalI and Eco RI, UCC53 with pHIS3 cut with Bam HI, UCC74 with VII-LURA3-TEL cut with Sal I and Eco RI, UCC75 with adh4::URA3 cut with BamHI and Sal I, and UCC76 with pUCU cut with Hind III. The expectedchromosome structure of each transformant was verified by Southernanalysis.

3. Selection for 5-FOA^(R) Colonies

Cells were grown into colonies for 2-3 days at 30° on YC plates orplates that lacked uracil. Colonies were picked and resuspended in 1.0ml H₂O, serial dilutions were made and an appropriate amount of cellsuspension was spread to produce ˜200 colonies/plate. Cells were spreadonto 5-FOA^(R) plates for selection, and YEPD, YC, or synthetic completemedia plates to determine the total number of colony forming cells. Thenumber of colonies on a plate was determined after 3-4 days of growth at30°.

4. Analysis of Nucleic Acids from S. Cerevisiae

S. cerevisiae cells were grown in 5 ml of YEPD to stationary phase, andtotal genomic DNA was isolated by disrupting cells with glass beads asdescribed (Runge and Zakian, 1989). Methods for cleavage of totalgenomic DNA with restriction enzymes, gel electrophoresis, and Southernhybridizations have been previously described (Gottschling and Cech,1984; Runge and Zakian, 1989). For rehybridization studies, probes wereremoved from blots with boiling water.

Cells were grown to a density of 0.5-2×10⁷ cells/ml and total RNA wasisolated as described (Sherman et al., 1986), except that the nucleicacids were precipitated with 2 vol. ethanol and resuspended in water ata concentration of 1-10 mg/ml. RNA concentration was determined by UVspectroscopy. Ten or twenty μg of total RNA was separated byelectrophoresis on a 1.5% agarose-formaldehyde-MOPS gel and transferredto nitrocellulose or nylon membrane as described (Ogden and Adams, 1987;Wahl et al., 1987). Strand specific RNA probes were made by in vitrotranscription of linearized plasmids with T7 polymerase in the presenceof [α-³²P] CTP (˜600 Ci/mmole) (Wahl et al., 1987).

Plasmids used for transcription were derivatives of pVZ1 (Eghtedarzadehand Henikoff, 1986); the URA3 probe contained the Pst I-Nco I fragmentof the gene, the HIS3 probe contained the Bam HI-Kpn I fragment of thegene, the TRP1 probe contained the Hind III-Bgl II fragment of the gene.Northern hybridization was performed as described (Wahl et al., 1987).Multiple exposures of autoradiograms were scanned with an LKB UltroscanXL densitometer to determine the relative levels of URA3 or HIS3 mRNA.

B. Results

1. Marking a Telomere with URA3

URA3, which is required for uracil biosynthesis, is normally found nearthe centromere on chromosome V. The entire gene, including the ciselements required for its normal regulation, is located on a 1.1 kbpHind III-Sma I fragment (Rose et al., 1984). This fragment was used inall of the URA3 constructs described in this example. Studies werecarried out in haploid yeast strains that contained either of twonon-reverting ura3⁻ alleles: ura3-52, which contains a Ty transposoninsertion within the URA3 coding sequence (Rose and Winston, 1984) (UCCseries), or ura3Δ::LEU2, in which the entire coding region of URA3 onchromosome V has been replaced by the LEU2 gene (DG series).

ADH4 is the most distal gene on the left arm of chromosome VII (Waltonet al., 1986). Fragment mediated transformation (Rothstein, 1983) wasused to introduce URA3 onto the left arm of chromosome VII, to createthe haploid strain DG28. In DG28, a portion of ADH4 and the DNA distalto it are deleted and replaced with URA3 and an 81 bp stretch of(TG₁₋₃). After transformation into yeast, the 81 bp are extended to ˜300bp of (TG₁₋₃), a length typical of all other telomeres in this strain.Transcription of the URA3 gene is towards the telomere, with itspromoter ˜1.3 kbp from the end of the chromosome.

2. Position Effect at Yeast Telomeres

The chemical 5-fluoro-orotic acid (5-FOA) can be used in the negativeselection of URA3 expression; 5-FOA is converted into a toxic substanceby the URA3 gene product (Boeke et al., 1987). The constitutive level ofURA3 expression in a cell is normally sufficient to yield cellssensitive to 5-FOA (5-FOA^(S)). Resistance to 5-FOA (5-FOA^(R)) can beused as a method to select for cells that have lost or mutated the wildtype URA3 gene. Therefore, sensitivity to 5-FOA was used as a means todetermine URA3 expression as a function of chromosomal location.

The frequency of a spontaneous 5-FOA^(R) allele arising at the normalURA3 locus is ˜10⁻⁷ (1GA2; Boeke et al., 1984). Since 5-FOA^(R) cellsderived in this way have mutations in the URA3 gene, they are Ura⁻ (i.e.unable to grow in the absence of uracil). In contrast, when cells withURA3 at the telomere (DG28) were pre-grown in media containing uracil(no selection for URA3 expression) and then plated for single coloniesonto 5-FOA, 33% of the cells gave rise to 5-FOA^(R) colonies (DG28).Moreover, when these 5-FOA^(R) colonies were replica-plated to mediathat lacked uracil, cells were able to grow. That is, the cells werestill URA3⁺. These results suggested that the 5-FOA^(R) exhibited bythese cells was not due to an inordinately high mutation rate in or lossof the URA3 gene, but rather that URA3 expression at the telomere wasreduced below the killing threshold of 5-FOA. Nonetheless, cells in an5-FOA^(R) colony still had the ability to produce sufficient URA3 geneproduct to overcome a lack of uracil in the medium.

When DG28 cells were pre-grown in medium lacking uracil (selecting forURA3 expression), one out of 10⁵ cells produced a colony on platescontaining 5-FOA. Once again, each of these 5-FOA^(R) colonies was stillURA3⁺. Taken together these results suggested that under non-selectivegrowth conditions expression of the URA3 gene in about one-third of theDG28 cells was sufficiently repressed to allow growth on 5-FOA, but thatunder selection, expression of the telomere-linked URA3 gene is stillpossible in many or all of the cells. The resistance to 5-FOA of cellswith URA3 at the VII-L telomere has been observed in a number ofstrains. While there have been strain specific differences in thefraction of 5-FOA^(R) cells when cells were pre-grown undernon-selective conditions, these values (0.10-0.90) were all within anorder of magnitude of one another (UCC74 & UCC81), and indicate thatrepression of a telomere-linked URA3 gene on VII-L is a generalphenomenon.

This unexpected behavior of the URA3 phenotype (colonies that were5-FOA^(R) yet still Ura⁺) caused the inventors to examine the steadystate levels of URA3 mRNA in cells with the gene either at its normalchromosomal position or at the telomere of VII-L. RNA was isolated fromcells grown under either selective or non-selective conditions for URA3expression. Consistent with earlier studies, cells with URA3 at itsnormal chromosomal locus had a modest increase in URA3 mRNA levels(˜1.4-fold) when grown under selective conditions compared to growthunder non-selective conditions (strain 1GA2) (Bach et al., 1979;Lacroute, 1968; Rose and Botstein, 1983). However, a major difference inURA3 mRNA levels was observed in cells with the URA3 gene at thetelomere. RNA levels in DG28 cells grown under non-selective conditionswere one-fifth that of cells with URA3 at its normal locus (DG28 &1GA2).

In contrast, under selective conditions, RNA levels in cells with URA3at the telomere were equivalent to the level in cells with URA3 at itsnormal locus (strains DG28 & 1GA2, INDUC). Thus consistent with the5-FOA^(R) phenotype, the constitutive level of URA3 RNA is significantlyreduced when the gene is located next to the telomere at VII-L comparedto when it is at its normal chromosomal locus. Yet under selection, thelevel of URA3 RNA at the telomeric locus in DG28 cells is virtually thesame as when URA3 is at its normal chromosomal locus.

In order to determine whether repression occurs at telomeres other thanVII-L, strain DG33 was constructed. This strain has the URA3 geneinserted near the telomere on the right arm of chromosome V (V-R), in amanner similar to that for URA3 on VII-L in strain DG28. Determinationof the fraction of 5-FOA^(R) colonies and analysis of mRNA levels instrain DG33 indicates that constitutive expression of URA3 is alsorepressed at this telomere. The difference in the fraction of 5-FOA^(R)cells between the two strains (0.33 for DG28, 0.04 for DG33) presumablyreflects differences between individual telomeres in terms of theirspecific chromosomal environments.

URA3 was also repressed when positioned near the telomere of atelocentric version of chromosome IV or of a 60 kbp artificial linearchromosome. Thus the ability to repress the expression of a nearby URA3gene appears to be a general property of S. cerevisiae telomeres.

3. Repression by Proximity to Telomeres Occurs for Other Genes

In general, a region of the chromosome that exerts a position effectdoes so in a gene non-specific manner. Therefore the inventors examinedwhether genes other than URA3 were repressed by proximity to a telomere.The TRP1, HIS3, or ADE2 gene was inserted between URA3 and the telomereDNA sequence at the Bam HI site of plasmid ‘VII-L URA3-TEL’. Each genewas inserted in both orientations. These constructs were then used tointroduce TRP1, HIS3, or ADE2 adjacent to the VII-L telomere, byselecting for URA3 expression. In selecting only for URA3 expressionduring strain construction, no selective pressure was placed upon thetelomeric TRP1, HIS3, or ADE2 genes.

In strains bearing a telomere-linked copy of TRP1, and grown undernon-selective conditions, TRP1 RNA was undetectable by Northernanalysis, regardless of the gene's orientation at the telomere (UCC61 &UCC62). By examining very long exposures of the autoradiograms theinventors estimated that the RNA level from the telomeric TRP1 was ≦1%of the RNA level when the same TRP1 fragment was located at an internalchromosomal site within the normal URA3 locus on chromosome V.

Colonies of cells with TRP1 at the telomere or at an internal locus weregrown on non-selective medium and then plated in serial dilution tomedium that lacked tryptophan. All of the cells with TRP1 at an internalsite on the chromosome (UCC63) formed colonies on plates lackingtryptophan. However, those with TRP1 at the telomere showed a reductionin colony forming ability on plates lacking tryptophan (UCC61 & UCC62).Only 10⁻² cells with TRP1 oriented such that transcription was directedtowards the telomere formed colonies in the absence of tryptophan(UCC61). When TRP1 transcription was away from the telomere, ˜10⁻³ cellsformed colonies (UCC62). In addition, the UCC61 cells formed robustcolonies in three days, while the UCC62 colonies were smaller.

The telomeric TRP1 RNA levels and plating efficiency data indicate thatunder non-selective growth conditions the majority of cells with TRP1near the telomere had very low or no TRP1 expression. In all three TRP1constructs described, a portion of the UAS/promoter elements found atthe normal TRP1 locus was missing (Kim et al., 1986). While thesemissing elements have no apparent effect on the ability of cells to growwithout tryptophan when TRP1 is at an internal locus, their absence mayexplain why TRP1 expression was more severely repressed at the telomerecompared to the expression of URA3 at the telomere.

When HIS3 was placed at the telomere and its transcription was directedaway from the telomere, there was a detectable reduction in RNA levelscompared to when the gene was at its normal chromosomal locus (UCC52).When the direction of transcription at the telomere was reversed, therewas a slight increase in RNA levels (UCC51). Phenotypically, there was amodest (less than ten-fold) reduction in plating efficiency on medialacking histidine for UCC52, but no effect on UCC51, a result consistentwith the relative RNA levels.

4. Transcriptional Repression at Telomeres is Reversible and Inheritedin a Semi-Stable Fashion

As shown above, when the URA3 gene was telomere-linked (DG28), cellsfrom colonies that were 5-FOA^(R) could still grow when placed on mediumlacking uracil. Conversely, cells grown in the absence of uracil wereable to form colonies when placed on medium containing 5-FOA. Theseresults suggested that a telomere-linked URA3 gene could switch betweenrepressed and active transcriptional states. The ADE2 gene provides aconvenient color assay for determining whether the gene is expressed;ADE2⁺ colonies are white, whereas ade2⁻ colonies are red (Roman, 1956).Thus, expression of a telomere-linked ADE2 gene can be monitored bydetermining the color of colonies produced by cells carrying this markedtelomere.

When the ADE2 gene was placed at the telomere such that ADE2transcription was directed towards the telomere (UCC41), all coloniescontained red and white sectors. This sectored phenotype indicated aswitch between the repressed and active transcriptional states of ADE2during colony development. The colonies displayed a wide range ofsectoring phenotypes. Some colonies were primarily white (active) andgave rise to red (repressed) sectors near the periphery. An equal numberof colonies were primarily red with white sectors near the periphery.Intermediate levels of sectoring between these two extremes were alsoreadily visible.

In some colonies multiple switches between transcriptional states can beinferred. For example, a predominantly red colony has a large whitesector. Within this white portion, red sectors are clearly visible. Thereversibility was further demonstrated by isolating cells within a white(or red) sector and plating them for single colonies. Each new colonycontained red and white sectors. In contrast with the results in UCC41,when ADE2 transcription was directed away from the telomere (UCC42), nored sectors were observed in a colony.

These results demonstrate that the expression of a telomere-linked ADE2gene can switch between an active and repressed state, and that theexpression state is semi-stable during mitotic growth. Based on theresults with URA3, TRP1, and HIS3, the inventors infer that the controlof ADE2 expression is at the transcriptional level. However, it was notpossible to determine the level of RNA produced at the telomere-linkedADE2, because an identical sized transcript was made by the ade2 alleleat its normal chromosomal locus.

The probability of a telomere-linked ADE2 gene (UCC41) being in anactive (repressed) transcriptional state was estimated from the fractionof predominantly white (red) colonies. Five colonies of UCC41 cellsgrown on non-selective medium, were plated for single colonies ontonon-selective medium. Approximately equal numbers of colonies were foundthat had primarily red centers giving rise to white sectors, andprimarily white centers that gave rise to red sectors. This resultindicates that a telomere-linked ADE2 gene on VII-L has an about equalprobability of being in an active or repressed transcriptional state.

However, when five colonies of UCC41 were pre-grown in the absence ofadenine (selecting for ADE2 expression) and then plated ontonon-selective plates, there were up to nine times as many colonies withwhite centers than with red centers. Closer examination of colonies withwhite centers revealed that red sectors generally did not appear untilvery close to the periphery of the colony. This observation suggestedthat the active expression state of ADE2 was stable for manygenerations. The distance from the center of these colonies to thepoints at which multiple red sectors appeared was measured. This valuewas used to compute the fraction of the total colony volume (assuming ahalf sphere geometry for a colony) that comprised the non-sectoredcenter of the colony. The number of cells within this region of thecolony was calculated (assuming there are ˜10⁸ cells in a colony), andthis value was used to derive the number of cell divisions required toproduce the quantity of white cells from a single progenitor. From thesecalculations the inventors estimate that the active transcriptionalstate of ADE2 is inherited for 15-20 generations in these colonies.

The phenotypic switching displayed by ADE2 at a telomere was alsoobserved with the URA3 gene using a single cell analysis. Freshly buddedcells grown on medium containing 5-FOA were moved by micromanipulationto a region of the plate where they could develop into full colonies.The majority (81/119) of the cells formed colonies, but 8% (9/119) ofthe cells formed microcolonies consisting of 4-8 cells. Microcolonieswere not detected in a control study in which no 5-FOA was present inthe medium. Therefore the microcolonies presumably represent cellsarrested in growth on the 5-FOA due to the URA3 gene being switched toan actively expressing state after budding. The cells that did not formcolonies may have been progeny of cells that had switched to an activelyexpressing state prior to budding, or were inviable as a result of themicromanipulation method. A rough value for the switching of URA3 from arepressed to an active state in DG28 cells was calculated by dividingthe number of cells that formed microcolonies (9) by the total number ofcolony forming cells (9+81), which yields an estimated switch rate of10⁻¹ per division.

5. The Distance Over which Telomeres Exert a Position Effect on URA3Expression

In order to obtain an estimate of the distance over which the telomereexerted a position effect, the URA3 gene was placed ˜20 kbp from the endof the left arm of chromosome VII by insertion within the ADH4 locus(DG27). Based on both RNA analysis and the frequency of 5-FOA^(R) colonyformation, cells with URA3 inserted within ADH4 had levels of URA3expression comparable to cells with URA3 at its normal locus, undereither selective or non-selective growth conditions. Thus on the leftarm of chromosome VII the telomeric repression was no longer detectablewhen URA3 was ˜20 kbp from the telomere.

In order to determine whether repression occurred over distances lessthan 20 kbp from the telomere, the constructs described above, in whichTRP1, HIS3, or ADE2 was inserted between URA3 and the telomere DNAsequence, were analyzed for URA3 expression. The inserted genesincreased the distance between URA3 and the telomere by 0.85, 1.8, or3.6 kbp, respectively.

Cells with each of the constructs were pre-grown in complete syntheticmedium, thus no selection for the expression of URA3 or the insertedgene was introduced. The cells were then plated to medium containing5-FOA and the fraction of 5-FOA^(R) colonies was determined. Theanalysis revealed that as the distance between URA3 and the telomere wasincreased, the level of repression decreased. For instance, insertion ofthe 0.85 kbp TRP1 fragment yielded 5-FOA^(R) colonies at a frequency of0.02-0.14 (UCC61 and UCC62), while insertion of the 3.6 kbp ADE2fragment yielded ≦10⁻⁵ 5-FOA^(R) cells (UCC41 and UCC42). However, thelevel of URA3 expression was also influenced by the orientation of theinserted DNA fragment.

This conclusion was best demonstrated by the result with the HIS3fragment: when transcription of the HIS3 gene was towards the telomere(UCC51), ˜10⁻⁴ cells were 5-FOA^(R); when HIS3 transcription was awayfrom the telomere (UCC52), 0.26 of the cells were 5-FOA^(R). Theorientation of TRP1 and ADE2 had smaller, but detectable effects on URA3expression. Thus further studies on the level of URA3 expression as afunction of distance from the telomere must take into account both thecomposition and orientation of the DNA sequences located between thetelomere and URA3.

6. Internal Tracts of (TG₁₋₃) Do Not Cause Repression, but they CanBecome Chromosome Ends and Consequently Cause Position Effect

Internal tracts of (TG₁₋₃) sequence occur naturally between the telomereassociated elements X and Y′ and between tandem Y′ elements (Chan andTye, 1983a; Chan and Tye, 1983b). Internal (TG₁₋₃) tracts range from 50to 130 bp in length (Walmsley et al., 1984). In order to determinewhether these internal (TG₁₋₃) sequences might also exert a positioneffect, 81 bp of (TG₁₋₃) were introduced adjacent to the telomeric sideof URA3, within the ADH4 locus (DG26).

RNA levels in these cells were somewhat higher than in cells with URA3at its normal locus or at the ADH4 locus without (TG₁₋₃) (DG26, DG27, &1GA2). This elevated transcription was true for both constitutive andinduced URA3 gene expression. These elevated mRNA levels are probablyexplained by an enhancer-like activity associated with (TG₁₋₃) repeatsequences when they are adjacent to a gene in a non-telomeric location(Runge and Zakian, 1990). Whatever the mechanism responsible forelevated expression, the internal tract of 81 bp of (TG₁₋₃) at the ADH4locus clearly does not cause repression of constitutive expression.These data demonstrate that (TG₁₋₃) sequences are not sufficient tocause position effect: the URA3 gene must be positioned near a telomere(or alternatively, near a (TG₁₋₃) tract >81 bp) in order fortranscription to be repressed.

Consistent with the high level of URA3 expression seen in the RNAanalysis, the fraction of 5-FOA^(R) colonies from cells with URA3 nextto the internal tract of (TG₁₋₃) and grown in uracil was ˜10⁻⁶ (DG26CONST). Although this value was low compared to cells with atelomere-linked copy of URA3, it is an order of magnitude greater thanthe fraction of 5-FOA^(R) colonies in cells with URA3 at its normallocus (strains 1GA2 & DG26). Replica-plating of the 5-FOA^(R) coloniesderived from DG26 cells revealed that they were all still Ura⁺ (incontrast to 5-FOA^(R) colonies arising from cells with URA3 at itsnormal locus, which were typically Ura⁻). This phenotype is identical tothat seen for the cells with URA3 at the telomere (i.e. DG28),suggesting that the internal (TG₁₋₃) sequences might have becometelomeric in those cells able to form colonies on 5-FOA.

This hypothesis was confirmed by Southern analysis. In four out of fourindependent isolates in which DG26 cells gave rise to 5-FOA^(R)colonies, the URA3 sequences were on a restriction fragment of the sizeexpected for a telomeric location. In addition, Southern hybridizationdemonstrated that sequences immediately distal to the internal (TG₁₋₃)tract were no longer detectable in the 5-FOA^(R) cells. These resultsshow that internal tracts of (TG₁₋₃) sequence can spontaneously becomechromosomal ends by a mechanism that results in the deletion ofsequences distal to the internal (TG₁₋₃) tract. In addition, the resultsprovide independent evidence that the repressed expression of URA3 atthe telomere is neither an artifact of transformation, nor a mutationwithin the URA3 gene or one of its trans activating factors.

C. Discussion

1. Position Effect at Yeast Telomeres

A position effect was demonstrated at the telomeres of S. cerevisiaechromosomes. The effect resulted in reduced gene expression oftelomere-linked genes as assayed both by amount of stable mRNA and byphenotype. For instance, cells with a telomere-linked URA3 gene wereable to grow in the presence of 5-FOA, behavior consistent with a ura3⁻phenotype. When ADE2 was telomere-linked many cells producedpredominantly red colonies as is characteristic of ade2⁻ cells. Theposition effect altered the expression of four out of four Pol II genes:ADE2, HIS3, TRP1, and URA3. In addition the effect was observed at fourout of four telomeres, including an artificial linear chromosome.Therefore, it can be concluded that the position effect is a generalphenomenon of S. cerevisiae telomeres.

The position effect acted upon the URA3 promoter at distances of atleast ˜4.9 kbp from the telomere, but at ˜20 kbp from the left end ofchromosome VII the effect was not observed. In addition the influence ofdistance on position effect strongly depended upon the specific DNAsequences located between URA3 and the telomere and probably otherfactors that are not yet well understood. For example, thetranscriptional activity of ADE2, HIS3, and URA3 was dependent upon thegene's orientation with respect to the telomere. In the process ofgenerating artificially fragmented linear chromosomes, Hegemann et al.report a “leaky” 5-FOA^(R) phenotype for a URA3 gene located 6-8 kbpfrom the telomere (Hegemann et al., 1988). In these constructs, most ofthe 6-8 kbp was the subtelomeric middle repetitive element Y′. Theinventors postulate that the reported “leaky” phenotype is due to atelomeric position effect and suggest that telomeric repression can actat a distance of at least 6 kbp, and through a Y′ element.

The telomeric position effect appears to be a result of proximity to theend of the chromosome and not simply due to the telomeric DNA sequence(TG₁₋₃). Eighty-one base pairs of (TG₁₋₃) sequence ˜20 kbp from thetelomere did not repress URA3 expression. While longer lengths of(TG₁₋₃) were not tested at internal loci, one of several strains thatwere tested for telomeric position effect contained the tell mutation.In the tell strain, the telomere adjacent to URA3 had a (TG₁₋₃) tract of95-120 bp, yet the level of 5-FOA^(R) in this strain was similar to thatfor all other strains tested. Taken together these results argue thatthe telomere itself, not simply (TG₁₋₃) repeats are responsible fortelomeric position effect in S. cerevisiae.

The repressed state conferred by the telomere was mitotically inheritedover a number of generations, but the genes could escape from repressionand switch to a state of active transcription. This reversibility wasvisually demonstrated by the red and white sectored colonies of cellswith ADE2 near the telomere (UCC41), and was also supported by thesingle cell analysis of DG28 cells on 5-FOA. The transcriptional stateof a gene, whether repressed or active, appeared to be stable over manygenerations.

The switching between active and repressed transcriptional states forgenes at telomeres is not due to genetic alteration, but rather to anepigenetic switch. Several lines of evidence support thisinterpretation: 1. The repression was readily reversible, in thepresence or absence of selection. 2. There were no differences in DNAstructure or copy number of the telomeric genes, as judged by Southernanalysis, regardless of whether these haploid cells were grown underconditions that were non-selective, or that selected for expression orrepression of the genes. 3. The telomeric position effect was genenon-specific.

Epigenetic variation of gene expression has been observed in plants,insects, mammals, and S. cerevisiae. In Drosophila, position effectvariegation is observed when a euchromatic gene is moved within or neara heterochromatic region of the chromosome (Eissenberg, 1989; Spofford,1976). Heterochromatin is a portion of the chromosome which remainsvisibly condensed throughout interphase of the cell cycle. In contrast,euchromatin decondenses after telophase and appears diffuse duringinterphase. When the white gene is located near some types ofheterochromatin, a ‘salt-and-pepper’ mosaicism in eye color is observed(Spofford, 1976). This mosaicism is visually analogous to the sectoredcolonies produced by cells with ADE2 at the telomere, and it could beinferred that similar mechanisms are at work in the two organisms.

In S. cerevisiae, epigenetic switching has been reported at the silentmating type locus, HML (Pillus and Rine, 1989). In a wild type cell HMLαis not expressed. However in a sir1 strain, HMLα switches betweenrepressed and expressed states. Current models for the HML switch favora change in chromatin conformation between the two phenotypic states.Besides changes in chromatin structure, postulated mechanisms ofepigenetic variation in plants and mammals include changes in DNAmethylation, topology, and nuclear locale (Pedoroff et al., 1989;Holliday, 1987; Monk, 1990; Weintraub, 1985).

Cytological observations in plants, insects, and mammals indicate thattelomeres occupy specific regions within the nucleus and are frequentlyassociated with the nuclear envelope (Lima-de-Faria, 1983a; White,1973). In addition, telomeres are usually packaged as heterochromatin(Fussell, 1975; Traverse and Pardue, 1989). In the single-celledeukaryotes, Oxytricha, Dictyostelium, and Tetrahymena, the DNA adjacentto the chromosome termini are packaged in an orderly array of phasednucleosomes, which is consistent with the presence of a highly orderedchromatin structure (Budarf and Blackburn, 1986; Edwards and Firtel,1984; Gottschling and Cech, 1984). In Drosophila, P element-mediatedtransposition of the white gene near the 3R telomere results in mosaicexpression of the gene, indicative of a position effect caused byproximity to the heterochromatin observed at this telomere (Hazelrigg etal., 1984; James et al., 1989; Levis et al., 1985).

It is noted that S. cerevisiae telomeres have two of the classicfeatures of heterochromatin: telomeres replicate late in S phase(McCarroll and Fangman, 1988), and as shown here, they exert positioneffect on the expression of nearby genes. The inventors propose that thephenotypic switching of telomere-linked genes in yeast is the result ofa competition between the formation of a stable active transcriptionalcomplex and the normal telomeric chromatin structure that prevents geneexpression. Such a chromatin structure must originate from the end ofthe chromosome. In Oxytricha the molecular ends of macronuclearmini-chromosomes are recognized by a heterodimeric protein complex(Gottschling and Zakian, 1986; Price and Cech, 1989). Similar proteinsin yeast may form a telomeric structure that is important inestablishing the position effect.

The semi-stable, reversible repression (or expression) at yeasttelomeres may be analogous to a primitive developmental switch. Whencells with a telomere-linked copy of ADE2 were pre-grown under selectionfor ADE2 expression, most (˜80%) subsequently gave rise to colonies ofprimarily white (transcriptionally active) cells under non-selectivegrowth conditions. The active transcriptional state of ADE2 can beinherited for at least 15-20 generations after removal of selection.This primitive control mechanism for gene expression may be exploited bysome organisms to allow developmentally controlled expression oftelomere-linked genes. In Trypanosomes, telomeres are the exclusivegenomic expression sites for surface antigen genes (reviewed in (Paysand Steinert, 1988). Many telomeres within a cell can carrytranscriptionally competent genes, yet only one such gene is expressedat a time. Perhaps the other telomere-linked genes are kept repressed,albeit reversibly, by telomeric position effect.

2. New Telomere Formation

The inventors found that internal tracts of (TG₁₋₃) sequence canspontaneously become chromosomal ends. Since the DNA distal to the(TG₁₋₃) tract is deleted, it seems unlikely that telomere formationoccurred by reciprocal recombination between the internal (TG₁₋₃)sequence and another telomere. New telomere formation may have occurredthrough intrachromosomal recombination between the internal (TG₁₋₃)sequence and the telomere with a resulting deletion of interveningsequences (as has been postulated for deletion of the subtelomericrepeat Y′ (Horowitz and Haber, 1985)), by unequal sister chromatidexchange or conversion, or by a distal chromosome break followed bytelomere “healing” at the (TG₁₋₃) sequence.

New telomere formation in conjunction with deletion of all terminalsequences has been observed cytologically, and has been an area ofintense interest because of its implications for chromosome breakage atfragile sites and for the generation of chromosomal abnormalities incancer cells (Le Beau, 1988; Sutherland and Hecht, 1985). Recently ithas been postulated that a subclass of such sites might in fact beregions of the chromosome which contain internal stretches of telomericDNA sequences (Hastie and Allshire, 1989). In this example the inventorsfind that internal tracts of telomeric DNA do indeed spontaneouslybecome chromosomal termini, albeit at a low frequency (˜10⁻⁶).

EXAMPLE II Modifiers of Position Effect are Shared Between Telomeric andSilent Mating-Type Loci in S. Cerevisiae

The inventors have shown that Pol II-transcribed genes succumb to aposition effect when placed near the ends of chromosomes in S.cerevisiae (Gottschling et al., 1990; Example I), reflectingobservations made in other eukaryotes that the chromosomal location of agene can affect its expression (Eissenberg, 1989; Henikoff, 1990;Lima-de-Faria, 1983; Spofford, 1976; Spradling and Karpen, 1990; Wilsonet al., 1990). The position effect is manifested as the stable butreversible transcriptional repression of each gene examined.

The mechanism by which this repression occurs is unclear, but it islikely due to a structural attribute of S. cerevisiae telomeres.Cytological observations in plants, insects, and mammals indicate thattelomeres are heterochromatic; in addition, the telomeres in theseorganisms and in Trypanosomes occupy unique locations within thenucleus, typically being associated with the nuclear envelope (Chung etal., 1990; Fussell, 1975; Hochstrasser et al., 1986; Lima-de-Faria,1983; Rawlins and Shaw, 1990; Traverse and Pardue, 1989; White, 1973).

HML and HMR are two other loci in S. cerevisiae where a position effecton transcription has been observed (Klar et al., 1981; Nasmyth et al.,1981). The mating-type genes, which are expressed when present at theMAT locus, are maintained transcriptionally silent when present at HMLand HMR even though all cis-acting sequences required for fullexpression at MAT are present. Other Pol II- or Pol III-transcribedgenes are also repressed when inserted within or near the HM loci (Brandet al., 1985; Mahoney and Broach, 1989; Schnell and Rine, 1986).

DNA sequences known as ‘silencers’ flank both HM loci and are requiredfor transcriptional repression (Abraham et al., 1984; Brand et al.,1985; Feldman et al., 1984; Mahoney and Broach, 1989). The silencers(denoted “E” and “I”) have been genetically dissected into smallerfunctional elements, which are recognition sites for DNA bindingproteins; these include an ARS (Autonomous Replicating Sequence)element, and ABF1 and RAP1 binding sites (Brand et al., 1987; Buchman etal., 1988; Mahoney and Broach, 1989; Mahoney et al., 1991; Shore andNasmyth, 1987; Shore et al., 1987). The RAP1 protein also binds to theyeast telomeric sequence (TG₁₋₃)_(n) (Buchman et al., 1988; Longtine etal., 1989). RAP1 is apparently involved in repression of HM, since HMRis derepressed when RAP1 temperature sensitive mutant cells are shiftedto the nonpermissive temperature (Kurtz and Shore, 1991).

At least seven additional genetic loci play a role in HM silencing. Theproducts of four genes, SIR1, SIR2 (MAR1), SIR3 (MAR2, CMT), and SIR4(Silent Information Regulator), are required for complete silencing atboth the HM loci (Haber and George, 1979; Hopper and Hall, 1975; Ivy etal., 1985; Ivy et al., 1986; Klar et al., 1979; Rine et al., 1979; Rineand Herskowitz, 1987). The molecular mechanism by which the SIR genesact to repress transcription is unclear; none of the SIR proteins havebeen demonstrated to bind silencer sequence DNA (Buchman et al., 1988;Shore et al., 1987).

A null allele of either NAT1 (N-terminal AcetylTransferase) or ARD1(ARrest Defective) causes several phenotypes, one of which isderepression of the silent mating type locus HML (Mullen et al., 1989;Whiteway et al., 1987). NAT1 and ARD1 appear to encode an N-terminalacetyltransferase, however it is not known whether the acetyltransferaseactivity acts directly in silencing at HML.

S. cerevisiae harbors two copies of genes encoding histone H4 (HHF1 andHHF2), either of which alone is sufficient for viability (Kim et al.,1988). In strains with deletions of HHF1 (hhf1::HIS3), single pointmutations in any of four consecutive amino acids (residues 16-19) nearthe N-terminus of histone H4 (HHF2) relieve transcriptional silencing atHML, with no other apparent phenotypic consequence (Johnson et al.,1990; Megee et al., 1990; Park and Szostak, 1990). These resultsdirectly implicate chromatin in HM silencing. Further evidence for theinvolvement of chromatin in silencing is suggested by theinaccessibility of HML and HMR to the HO endonuclease in vivo (Strathernet al., 1982; Kostriken et al., 1983). Additionally, in vitro nucleasesensitivity analysis of HML and HMR suggests that the HM loci exist in adistinct chromatin structure that is refractory to transcription in aSIR dependent manner (Nasmyth, 1982).

The characteristics of position effect and RAP1 binding sites shared bytelomeres and the HM loci prompted the inventors to test whether theSIR, HHF2, NAT1, and ARD1 genes play a role in transcriptionalrepression at yeast telomeres. The results of this Example show that inaddition to their roles in silencing at the HM loci, the SIR2, SIR3,SIR4, NAT1, ARD1, and HHF2 genes are required for the telomeric positioneffect in S. cerevisiae.

Mutations in any of these genes relieves transcriptional repression ofeither URA3 or ADE2 at two different telomeres. In contrast, mutationsin SIR1 did not alter repression at telomeres. These results suggestthat telomeres in S. cerevisiae exist in a heterochromatin-likestructure; a structure composed of proteins which also function atsimilar chromosomal domains such as the HM loci. Based on thedifferences in silencing between telomeres, HML, and HMR, the inventorssuggest a hierarchy of chromosomal silencing exists within the yeastgenome.

A. Materials and Methods

1. Plasmid Constructions

Plasmid pADE2 contains the ADE2 gene on a 3.6 kbp chromosomal BamHIfragment from plasmid pL909 (obtained from R. Keil). Plasmid pΔADE2 wasconstructed by replacing the internal 2.2 kbp HindIII fragment (containsall but the six C-terminal residues of the ADE2 open reading frame;(Stotz and Linder, 1990) of plasmid pADE2 with the 3.8 kbp BamHI-BglIIfragment of pNKY51 which contains two direct repeats of the SalmonellahisG gene flanking URA3 (Alani et al., 1987). The HindIII and BamHIends, and the HindIII and BglII ends were blunt-ended with T4 DNApolymerase and ligated together, resulting in the destruction of theseparticular restriction sites Thus, pAADE2 contains a 5.2 kbp BamHIfragment with about 700 bp of homology to sequences upstream anddownstream of the ADE2 gene flanking the 3.8 kbp BamHI-BglII(hisG-URA3-hisG) fragment from pNKY51.

A 2.4 kbp HindIII fragment from plasmid pJR104 (obtained from J. Rine)which contains the 5′ end of the SIR3 gene was inserted into pVZ1 toyield plasmid pH3SIR3. Plasmid pH3SIR3 was digested with BglII to excisea 600 bp fragment in the SIR3 coding sequence, which was replaced with a1.8 kbp BamH1 fragment containing the HIS3 gene. The resulting plasmidwas pΔSIR3::HIS3.

2. Yeast Strains and Methods

Media used for the growth of S. cerevisiae were described previously(Gottschling et al., 1990; Example I). S. cerevisiae were transformed bythe lithium acetate procedure (Ito et al., 1983) or by electroporationin the presence of sorbitol (Becker and Guarente, 1991).

The URA3 gene was placed adjacent to the telomere sequence (TG₁₋₃)_(n)on the left end of chromosome VII (UCC1-UCC5, UCC16, UCC18, UCC25,UCC128, UCC2031-UCC2036), or the right end of chromosome V(UCC31-UCC35); no telomere associated sequences (i.e.: X and Y′ elements(Chan and Tye, 1983a; Chan and Tye, 1983b)) were present. Alternatively,the ura3-52 or ura3-1 allele (at the normal URA3 locus on chromosome Vin the parent strains) was converted to URA3⁺ (UCC6-UCC10, and UCC129),or URA3 was inserted into the ADH4 locus about 20 kbp from the telomereon VII-L (UCC11-UCC15).

Strains UCC5, UCC6, UCC12, and UCC35 were derived from DBY703; UCC1,UCC7, UCC11, and UCC31 were derived from JRY1705; UCC2, UCC8, UCC13, andUCC32 were derived from JRY1706; UCC3, UCC9, UCC14, and UCC33 werederived from JRY1264; UCC4, UCC10, UCC15, and UCC34 were derived fromJRY1263. Strain UCC18 was derived from W303-1a; UCC16 was derived fromAMR1; UCC25 was derived from JRM5. UCC128 and UCC129 were derived fromYDS73; strain UCC2031 was derived from LJY153, UCC2032 from LJY405I,UCC2033 from LJY412I, UCC2034 from LJY421I, UCC2035 from LJY305TR1,UCC2036 from LJY305T. Plasmids and methods for these constructions aredescribed in Example I and Gottschling et al. (1990).

Strains UCC46 (SIR⁺), UCC47 (sir1), and UCC48 (sir4), which were derivedfrom strains DBY703, JRY1705, and JRY 1263, respectively, harbor anade2Δ. The ade2Δ was made by transformation of strains DBY703, JRY1705,and JRY1263 with plasmid pAADE2 digested with BamHI, followed byselection for URA⁺ transformants. In these transformants the ADE2 openreading frame has been replaced (all but the six C-terminal residueswere deleted) with a DNA fragment containing two direct repeats of theSalmonella hisG gene flanking URA3. Loss of URA3 by recombinationbetween the two hisG repeats within the ade2 locus was screened for by5-FOAR (Alani et al., 1987).

Strains UCC84, UCC86, and UCC88, derived from UCC46, UCC47, and UCC48,respectively, and strains UCC97, UCC98 and UCC99, derived bytransformation of strains W303-1a, AMR1, and JRM5, respectively, have afunctional ADE2 gene located adjacent to the chromosome VII-L telomere(ADE2-TEL) (Example I); no telomere associated sequences (i.e.: X and Y′elements (Chan and Tye, 1983a; Chan and Tye, 1983b)) were present.Strains UCC2037-UCC2042, derived from strains LJY153, LJY405I, LJY412I,LJY421I, LJY305T, and LJY305TR1, respectively, were constructed in thesame manner to place ADE2 adjacent to telomere VII-L.

Strain UCC121 was derived from W303-1a by transformation with a 3.6 kbpBamH1 ADE2⁺ fragment and selection for ADE⁺ transformants. Strain UCC120was constructed by introduction of plasmid pJR531 (Kimmerly and Rine,1987) which had been digested with SphI and EcoRV into UCC97, andselection for HIS⁺ transformants. Strain UCC131 was constructed byintroduction of pΔSIR3::HIS3 which had been digested with EcoRI intoUCC84, and selection for HIS⁺ transformants.

Strains UCC122-UCC125, UCC138, and UCC139 were constructed bytransformation of strains UCC16, UCC18, UCC19, UCC21, UCC128, andUCC129, respectively, with plasmid PKL1. Plasmid PKL1 contains the SIR1gene on a 2μ-based vector which contains TRP1 for selection (Stone etal., 1991).

The expected structures of the various chromosomal constructs wereconfirmed by gel electrophoresis followed by DNA blot hybridizationanalyses. The sir⁻ phenotypes of strains UCC120 and UCC131 wereconfirmed by their inability to mate (Sprague, 1991).

3. Quantification of 5-FOA Resistance Cells from isolated colonies grownon rich medium for 2-3 days at 30° were inoculated into liquid mediumcontaining (100 mg/L) uracil. When these cultures reached mid-log phase,serial dilutions were plated onto synthetic complete medium or mediumcontaining 5-FOA (Example I; Gottschling et al., 1990). 5-FOA resistancewas determined as the average ratio of colonies formed on 5-FOA mediumto colonies formed on complete medium, from a minimum of threeindependent trials, using different colony isolates for each trial. Thenumber of colonies on a plate was determined after 3-4 days of growth at30° C. Alternatively, colonies of strains grown on rich medium two tothree days were suspended in H₂O, and ten-fold serial dilutions wereplated as described above. For some strains, selection for TRP⁺ wasrequired to maintain episomal plasmids; these strains were grown onsynthetic medium lacking tryptophan three to four days and colonies weresuspended in H₂O, serially diluted, and plated as above on syntheticmedium lacking tryptophan or on 5-FOA medium lacking tryptophan.

4. Analyses of Nucleic Acids from S. Cerevisiae

Preparation and analyses of nucleic acids were as in Example I, exceptthat some DNA blot hybridization analyses were carried out using theGenius system from Boehringer Mannheim following the manufacturer'sprocedures.

B. Results

1. SIR2, SIR3, and SIR4 Maintain Transcriptional Repression at Telomeres

An isogenic set of sir⁻ strains with the URA3 gene located at one offour different chromosomal sites was constructed: adjacent to telomereVII-L or V-R, at its normal chromosomal location, or at a secondnon-telomeric site (ADH4, ˜20 kbp from telomere VII-L). URA3 expressionwas measured by two criteria: resistance to 5-fluoroorotic acid(5-FOA^(R)), and URA3 mRNA levels as determined by RNA blothybridization analysis. 5-FOA is converted into a toxic metabolite bythe URA3 gene product, such that cells expressing normal levels of theURA3 gene product are killed on media containing 5-FOA, whereas ura3⁻cells are resistant to 5-FOA (5-FOA^(R)) (Boeke et al., 1987). Cellswith URA3 near a telomere form colonies on 5-FOA medium, yet cellswithin these 5-FOA^(R) colonies can grow in the absence of uracil,indicating that genetically identical cells can switch from a clonallyinherited repressed state to a transcriptionally active state(Gottschling et al., 1990).

Consistent with these earlier results, when the URA3 gene was locatedadjacent to either the VII-L or V-R telomere in a SIR⁺ strain, asignificant fraction of cells were resistant to 5-FOA (0.62 for UCC5,0.15 for UCC35), and cells from 5-FOA^(R) colonies retained the abilityto form colonies on medium lacking uracil. Similar results were obtainedwith the sir1 strain, indicating that expression of the telomeric URA3gene is repressed in a subset of cells in these strains, and that theSIR1 gene product is not required for repression.

In contrast, a telomeric URA3 gene was not repressed in cells that weresir2, sir3, or sir4. The frequency of 5-FOA^(R) colonies arising fromthese strains (˜10⁻⁷) was equivalent to that seen for all strains withURA3 at its normal chromosomal locus or at the ADH4 locus. Mutations inthe SIR genes had no effect on the 5-FOA resistance of cells having URA3at either of these non-telomeric loci.

RNA blot hybridization analysis shows that sensitivity to 5-FOA as aresult of the sir mutations was a reflection of mRNA levels from thetelomeric URA3 gene. No URA3 mRNA was detectable in SIR⁺ or sir1 strainswhich had URA3 at the telomere and were grown under non-selectiveconditions (“uracil +”), even when the autoradiograph was greatlyoverexposed. URA3 mRNA was only detectable in the SIR⁺ or sir1 strainswhen they were grown to select for telomeric URA3 expression (“uracil−”), though this level was significantly lower than when URA3 was at itsnormal chromosomal locus.

In sharp contrast, the telomeric URA3 gene produced high levels of mRNAin sir2, sir3, and sir4 strains. These levels were comparable to thosefrom URA3 at its normal chromosomal locus. The sir mutations had noeffect on URA3 expression at its normal chromosomal locus or wheninserted within the ADH4 locus. These data indicate that the telomericposition effect on URA3 expression mediated by SIR2, SIR3, and SIR4 isat the level of transcription.

To demonstrate that the SIR requirement for the telomeric positioneffect was not gene specific, sir strains were constructed with the ADE2gene located at the VII-L telomere, or at its normal locus. The ADE2gene provides a visual color assay for its expression; ADE2⁺ strainsform white colonies, while ade2⁻ strains form red colonies (Roman,1956). Example I shows that a SIR⁺ strain containing a single copy ofADE2 at a telomeric locus exhibited phenotypic variegation of ADE2,manifested as red-and-white sectored colonies. Here it was found thatstrains with the telomeric ADE2 that were SIR⁺ or sir1 formed red andwhite variegated colonies, indicating that ADE2 was repressed in asubset of the cells within these colonies. The sir2, sir3, and sir4strains formed entirely white colonies, demonstrating that the telomericADE2 gene was not repressed (for sir2 and sir3). These results confirmthat the SIR2, SIR3, and SIR4 genes are required for maintainingtranscriptional repression at telomeres, in addition to silencing the HMloci (Rine and Herskowitz, 1987).

2. Single Point Mutations in Histone H4 Relieve TranscriptionalRepression at Telomeres

Single point mutations in any of four consecutive amino acids (residues16-19) near the N-terminus of histone H4 (HHF2) relieve transcriptionalsilencing at HML (Johnson et al., 1990; Megee et al., 1990; Park andSzostak, 1990). URA3 or ADE2 was placed at the VII-L telomere inisogenic strains that carried a single copy of either the wild-typehistone H4 (HHF2), or a mutated copy of HHF2. Three such pointsubstitution mutations, all of which derepress HML, were tested: achange of lys-16 to either gly-16 or gln-16, and a change of arg-17 togly-17.

Each strain that contained one point mutation in histone H4 exhibitedderepression of telomeric URA3 transcription as shown by theirinviability on 5-FOA. When ADE2 was near the telomere in strains withthese same histone H4 mutations, colonies were completely white, onceagain indicating derepression of the telomeric gene. Thus single pointmutations at residues 16 or 17 in histone H4 which replace the wild-typebasic amino acid with an uncharged residue, result in relief of thetelomeric position effect.

There is genetic evidence that SIR3 interacts with histone H4 to silencegenes at HML (Johnson et al., 1990). Alleles of sir3 (e.g. sir3R1) havebeen identified that can partially suppress the HML silencing defectcaused by certain point mutations in histone H4 (e.g.: lys-16 togly-16). URA3 was introduced at the VII-L telomere in an isogenic pairof strains which were either HHF2-gly16, SIR3⁺ (UCC2036) or HHF2-gly16,sir3R1 (UCC2035). No suppression by sir3R1 was observed at the telomereas judged by complete sensitivity to 5-FOA. Equivalent strains with ADE2at the telomere produced no red sectored colonies, supporting theconclusion that the sir3R1 allele could not restore repression at thetelomere in an HHF2-gly16 strain.

3. NAT1 and ARD1 are Required for the Telomeric Position Effect

A null mutation of either NAT1 or ARD1 causes derepression of the silentmating-type locus HML (Mullen et al., 1989; Whiteway et al., 1987). URA3or ADE2 was introduced at the VII-L telomere into each member of a setof isogenic strains that was either nat1, ard1, or wild-type for bothgenes. The sensitivity to 5-FOA of nat1 and ard1 strains was equivalentto that observed for sir2, sir3, and sir4 and the point mutants inhistone H4. Thus no position effect was observed for a telomeric URA3gene in nat1 or ard1 cells. Likewise, the telomeric ADE2 gene was notrepressed in the nat1 and ard1 strains as these strains formed entirelywhite colonies.

4. Overexpression Of SIR1 does not Restore Position Effect at Telomeres

Overexpression of SIR1 partially suppresses the mating defects of MATastrains containing nat1 or ard1 mutations, or certain sir3 or HHF2alleles by re-establishing silencing at HMLα (Stone et al., 1991). Theinventors tested whether SIR1 overexpression could restore silencing ofa telomere-linked gene in a nat1 or sir3::LEU2 strain. Plasmid pKL1(Stone et al., 1991) which contains SIR1 on a 2μ-based vector wastransformed into strains which were nat1, sir3, or wild-type and haveURA3 located at telomere VII-L or at the normal URA3 locus. As expected,a significant fraction of cells of strain UCC123 (wild-type,URA3-TEL/pKL1) were resistant to 5-FOA. However, the nat1 and sir3strains which have URA3 at telomere VII-L and harbor pKL1 continue to besensitive to 5-FOA, as are the strains with URA3 at its normalchromosomal locus. Thus the overexpression of SIR1 does not restoresilencing at telomeric loci in nat1 or sir3 strains These results areconsistent with the results presented above, indicating that SIR1 playsno role in transcriptional silencing at telomeres.

C. Discussion

1. Similarities and Differences in Position Effects at Telomeres and theHM loci

This example shows that the SIR2, SIR3, SIR4, HHF2, NAT1, and ARD1 genesare required for the position effect at telomeres in S. cerevisiae.Consequently, it implies that these gene products constitute a generalmechanism for silencing chromosomal domains in S. cerevisiae. In view ofthe results presented here, it is interesting to note that both HML andHMR are located quite close to the termini of chromosome III, ˜12 kbp(Button and Astell, 1986) and ˜25 kbp (Yoshikawa and Isono, 1990),respectively. When HML is present on a circular plasmid or a ringchromosome III derivative, deletion of HMLE or HMLI results inderepression of HML (Feldman et al., 1984; Strathern et al., 1979).However, these mutated HML loci are fully silenced when present at thenormal telomeric HML locus (Mahoney and Broach, 1989) suggesting theproximity of HML to the telomere may facilitate full repression of thislocus.

There was no detectable change in the telomere-specific position effectin sir1 strains or in strains with SIR1 on a high copy plasmid. Sinceboth of these genotypes have an effect on HML and HMR, the inventorsconclude that SIR1 function is specific to silencing of the HM loci.Single-cell analysis of sir1 strains indicates that a mixed populationof cells exists with ˜20% of cells being transcriptionally silent at HMLand the remainder being transcriptionally active at HML; thetranscriptional state is clonally inherited, though cells switch betweentranscriptionally active and repressed states at a low frequency (Pillusand Rine, 1989).

The inventors have found that epigenetic switching betweentranscriptional states occurs at telomeres in SIR⁺ (and sir1) strains,analogous to that observed at HML in sir1 mutants (Example I; Pillus andRine, 1989). The inventors therefore propose that SIR1 provides completesilencing at HML and HMR by preventing switching from the silent to theactive transcriptional state. The HM loci is thus proposed to containelements through which SIR1 acts, which are absent from chromosomaltermini (e.g.: the A and B elements (Brand et al., 1987)). In support ofthis, a recent study has identified deletions at HMLE which result inepigenetic switching of transcriptional states at HML (Mahoney et al.,1991).

A number of differences have been observed between silencing attelomeres, HML, and HMR, which may yield insights into the functionalorganization of the silent loci. As indicated above, the epigeneticswitching of HML expression in sir1 strains is very similar to theexpression of a telomeric gene in a SIR⁺ (or sir1) strain, indicatingthat elements through which SIR1 can act to fully silence HML arepresent at HML (and probably HMR) but not at telomeres. Also, while asir1 mutation has only a slight effect at either HM locus, and amutation in nat1 alone derepresses HML but not HMR (Mullen et al.,1989), the sir1, nat1 double mutant is completely derepressed at HMR,suggesting that additional mechanisms of silencing exist at HMR comparedto HML (or telomeres) (Stone et al., 1991). Deletion of NAT1 or ARD1results in significant derepression of HML but not HMR (Whiteway et al.,1987); however, deletion of the RAP1 binding site at HMRE results inderepression of HMR in nat1 or ard1 strains (Stone et al., 1991), againindicating that redundant silencing mechanisms exist at HMR compared toHML and telomeres.

Lastly, sir3R1 partially restores HML silencing in a HHF2-gly16 strain(mating efficiency is restored from ˜10⁻⁵ to ˜10⁻¹; (Johnson et al.,1990)), but does not restore telomeric silencing. This may be explainedif suppression of HHF2-gly16 by sir3R1 is facilitated by the presence ofa redundant silencing mechanism(s), such as that mediated by SIR1. Thusthe inventors suggest that telomeres exhibit a basal level oftranscriptional repression, and that silencing at HML and HMR is basedon the same mechanism(s), but is strengthened and regulated by thepresence of additional silencer elements.

2. How does the Telomeric Position Effect Occur?

Little is known about the specific mechanism by which the SIR, HHF2,NAT1, and ARD1 gene products act in transcriptional silencing, howeverthe available evidence suggests that they modify chromatin structure(Nasmyth, 1982). Single point mutations in histone H4 completely relievethe telomeric position effect and thus provide the best evidence thatchromatin structure is intimately involved in telomeric silencing.Mutations in any of four contiguous amino acids (residues 16-19) in theN-terminus of histone H4 result in derepression at HML (Johnson et al.,1990; Megee et al., 1990; Park and Szostak, 1990); these four positivelycharged amino acids are conserved throughout eukaryotes, and are sitesof post-translational modifications (van Holde, 1989). Significantly,correlative studies note that the modifications (e.g. acetylation andphosphorylation) on histone H4 are associated with the transcriptionalstatus of the chromatin (van Holde, 1989).

In yeast, suppressors of the histone H4 point mutations, which restoresilencing, map as compensatory changes in the SIR3 gene, thus providingevidence that SIR3 interacts with chromatin (Johnson et al., 1990). Inaddition, SIR2 has been shown to suppress intrachromosomal recombinationbetween rDNA repeats, supporting the idea that SIR2 may play a generalrole in chromatin organization (Gottlieb and Esposito, 1989).

NAT1 and ARD1 apparently encode two subunits of a yeast N-terminalacetyltransferase which acetylates histone H2B along with at leasttwenty other proteins (Mullen et al., 1989) which may play a direct rolein silencing by acetylation of H2B.

It has been reported that SIR4 shares sequence similarity with thecoiled-coil domains of human nuclear lamins A and C (Diffley andStillman, 1989). These domains facilitate polymerization of lamins intothe lamina, which lines the nuclear envelope. Taking into account thecytological observations in interphase nuclei which indicate telomeresare located at the nuclear periphery it is plausible that the putativepolymerization domain of SIR4 is associated with the nuclear lamina andmight therefore mediate binding of telomeres to the nuclear envelope.Since the SIR4 gene product is believed not to bind DNA directly(Buchman et al., 1988; Shore et al., 1987), an interaction between SIR4and a telomere binding protein (e.g. RAP1) may enable an associationbetween telomeres and the nuclear envelope. It is noteworthy thatpurified mammalian nuclear lamins A and C bind in vitro to syntheticoligonucleotides containing mammalian telomere repeat sequences (Shoemanand Traub, 1990). Thus attachment of telomeres, as well as otherchromosomal loci or regions, to the nuclear envelope may be a componentof nuclear organization, and might therefore affect local geneexpression (Alberts et al., 1989; Blobel, 1985).

The position effect at S. cerevisiae telomeres may reflect a generalfeature of eukaryotic telomeres. In Drosophila, stable transposition ofthe white gene to a position near a telomere results in a mottled eyecolor phenotype (Levis et al., 1985), which is consistent withtranscriptional repression of white in some cells. Cytological studiesin a number of organisms indicate that telomeres are organized intoheterochromatin (Lima-de-Faria, 1983; Traverse and Pardue, 1989). Whileheterochromatin is defined cytologically as a region of the chromosomewhich remains condensed in interphase, it also displays two distinctivetraits: late DNA replication, and the ability to repress transcriptionof euchromatic genes placed nearby (Eissenberg, 1989; Henikoff, 1990;Spofford, 1976; Spradling and Karpen, 1990). S. cerevisiae telomerespossess both of these hallmarks of heterochromatin (Example I; McCarrolland Fangman, 1988). The SIR2, SIR3, SIR4, HHF2, NAT1, and ARD1 productsmay be intimately involved with the organization of regions of yeastchromosomes into heterochromatin or heterochromatin-like structures.Because telomeres and histones are highly conserved structurally andfunctionally among eukaryotes, it seems quite likely that the mechanismof transcriptional repression functioning in S. cerevisiae is alsoutilized in multi-cellular eukaryotes.

EXAMPLE III Silent Domains are Assembled Continuously from the Telomereand are Defined by Promoter Distance and Strength and SIR3 Dosage

The eukaryotic genome is organized into regions distinct in theirstructure and function. Heterochromatin, which defines one suchstructural region, is condensed throughout the cell cycle, while itscounterpart, euchromatin, is more diffuse in appearance duringinterphase (Heitz, 1928, as cited in Brown, 1966). Chromosomal regionsalso differ functionally since the expression of a eukaryotic gene canbe profoundly affected by its chromosomal position. This phenomenon,chromosomal position effect, is observed in many eukaryotes(Lima-de-Faria, 1983) and has been extensively studied in Drosophilamelanogaster (Lewis, 1950; Baker, 1968; Spofford, 1976). When geneticrearrangements place euchromatic segments of the genome into or nearheterochromatin, the expression of a translocated euchromatic gene isaltered in a population of cells: some cells express the gene, whileothers do not. Thus a mosaic or variegated phenotypic pattern isproduced.

Chromosomal position effects phenomena can spread over great distancesin the genome; e.g., in Drosophila, genes located as far away as 80chromosome polytene bands (˜2000 kbp) are still subject toposition-effect variegation (PEV) (Demerec, 1940). This spreading effectis thought to reflect the dynamic nature of assembly of heterochromatinover a locus (Zuckerkandl, 1974; Spofford, 1976). When heterochromatinassembles far enough to include a locus, the gene within it isinactivated.

In Saccharomyces cerevisiae, chromosomal domains have been identifiedthat exert position effect: the cryptic mating-type loci, HML and HMR,and telomeres (Laurenson and Rine, 1992; Sandell and Zakian, 1992).Genes located near or within these domains may be transcriptionallysilenced and exhibit phenotypic variegation (Klar et al., 1981; Nasmyth,et al. 1981; Schnell and Rine, 1986; Mahoney and Broach, 1989; ExampleI). At least six modifiers of position effect are shared between the HMloci and telomeres. A mutation in SIR2, SIR3, SIR4, NAT1, ARD1, or HHF2(which encodes histone H4) reduces or abolishes silencing at telomeres,HML, and HMR (Hopper and Hall, 1975; Haber and George, 1979; Klar etal., 1979; Klar et al., 1981; Ivy et al., 1986; Rine and Herskowitz,1987; Whiteway et al., 1987; Kayne et al., 1988; Mullen et al., 1989;Megee et al., 1990; Park and Szostak, 1990; Example II; Aparicio et al.,1991).

The involvement of histone H4, and the observation that the HM loci andtelomeres are refractory to DNA modifications in vivo in a SIR-dependentmanner, point to chromatin structure as being involved in silencing theHM loci and telomeres. Specifically, this chromatin structure is thoughtto hinder access of transcription factors to these loci (Nasmyth, 1982;Kostriken et al., 1983; Klar et al., 1984; Gottschling, 1992; Singh andKlar, 1992).

Spreading of position effect also occurs in yeast (Abraham et al., 1984;Feldman, et al. 1984). Genes located up to ˜4.9 kbp from a telomerestill are subject to position effect, whereas no silencing is detectedat loci ˜20 kbp from the chromosome end (Gottschling et al., 1990).Additionally, insertion of a 30 kbp Ty-array between the E and I sites(cis-elements required for silencing) at HMLa relieves silencing at thislocus. However silencing is re-established when this array is reduced toa single 7 kbp Ty (Mastrangelo et al., 1992). Thus there is a limit tothe size of silenced domains at both HM loci and telomeres.

Telomeric silencing in yeast provides an excellent opportunity to studythe spread of position effect in a eukaryote, particularly because theinitiation site of position effect is known to be the end of thechromosome (Example I). In this Example, a quantitative method toexamine telomeric position effect was used to identify parameters thatmodulate spreading. The results provide a molecular and mechanisticinsight into the propagation of silencing in yeast, as well as thefunctional organization of silent chromosomal domains.

A. Methods

1. Construction of Plasmids

The set of plasmids used to insert the URA3 gene at various positionsalong V-R was constructed as follows, starting with plasmid pB610H(obtained from C. Newlon). Plasmid pHSS6TG carries a telomeric repeatsequence (derived from pYTCA-2; Example I) inserted between the EcoRIand BamHI restriction sites of plasmid pHSS6 (Seifert et al., 1986).Orientation of the telomeric sequence is such that digestion of pHSS6TGwith EcoRI will yield an end that is a substrate for telomere formationin yeast. A 7.3 kbp BamHI fragment from plasmid pB610H was ligated intothe BamHI site of pHSS6TG. Next, a 7.4 kbp NotI fragment of this newplasmid, carrying unique V-R sequences adjacent to a telomeric(TG₁₋₃)_(n) repeat, was cloned into the NotI site of pVZ1 (Henikoff andEghtedarzadeh, 1987), generating pSC1. Plasmids pVURAH2(+) andpVRURAH2(−) were constructed by inserting a 1.2 kbp HindIII fragmentcontaining URA3 into the “H₂” site of pSC1 partially digested withHindIII. URA3 transcriptional orientation is denoted (+) whentranscription is toward the telomere and (−) when toward the centromere.URA3 was cloned in a similar way into the “H₃” and “H₄” HindIIIrestriction sites, generating plasmids pVURAH3(+), pVURAH3(−),pVURAH4(+) and pVURAH4(−), respectively.

The HIS3 gene was isolated from plasmid pHIS3 (Struhl, 1985; Example I)by amplification using the polymerase chain reaction (Innis et al.,1990), using the following primers: 5′ oligo 5′CCGGATCCTGCCTCGGTAATGATTTCATTTTTT 3′ (SEQ ID NO:13); 3′ oligo 5′CCGGATCCTCTCGAGTTCAAGAGAAAAAAAAAGAAA 3′ (SEQ ID NO:14). Restrictionsites for BamHI, which were placed at the ends of the oligonucleotidesfor convenient cloning, are underlined. Hence, the inventors refer tothis DNA segment as “HIS3 BamHI fragment”.

Plasmids used to test for discontinuity of silenced chromosomal domainsalong V-R were created as follows: pH1.5HIS3(+) and pH1.5HIS3(−) wereconstructed in two steps. First, a 1.5 kbp HindIII fragment of V-Rchromosomal DNA was inserted into the HindIII site of pHSS6 to generateplasmid pHSS6(1.5). pHSS6(1.5) was then digested with KpnI, blunt-ended,and ligated with the HIS3 BamHI fragment which had its ends filled-in. Atwo-step process was also required to construct plasmids pVRUH2(−)HR1(+)and pVRUH2(−)HR1(−). Plasmid pVURAH2(−) was cut with XhoI and SalI, andrecircularized by ligation; a blunt-ended HIS3 BamHI fragment wasligated into this plasmid which had been partially digested with EcoRIand blunted with T4 DNA polymerase. Plasmids pVRUH2(+)HR1(+) andpVRUH2(+)HR1(−) were constructed following the same procedure. PlasmidspYAHIS4-2(−) were made by cloning the HIS3 BamHI fragment into the BamHIsite of pYA4-2 (Walton et al., 1986).

Plasmid pDPPR1-HIS3 was constructed by replacing a 0.7 kbp BglIIfragment containing the promoter region of PPR1 (Kammerer et al., 1984),in plasmid pUC8-PPR1 (obtained from R. Losson), with a 1.85 kbp BamHIfragment from plasmid pHIS3. In plasmid pDPPR1::LYS2 the same BglIIfragment was replaced by a blunt-ended 4.8 kbp HindIII-XbaI fragmentcontaining LYS2, isolated from pDP6 (Fleig et al., 1986).

Plasmid pVZ1DGCN4::TRP1 carries a deletion in the translation initiationregion of GCN4. Plasmid pB238 (a derivative of plasmid p164 (Hinnebusch,1985)) was digested with BamHI and BglII, and a 0.8 kbp BamHI fragmentcontaining TRP1 from YDpW (Berben et al., 1991) was ligated into it. ASalI-EcoRI 3.2 kbp fragment of the resulting plasmid was then ligatedinto pVZ1 previously digested with EcoRI and SalI, to createpVZ1DGCN4::TRP1.

The plasmid pVZJL38TRP1(+)ADE2(−) was used to insert TRP1 and ADE2between ADH4 and telomere VII-L. Plasmid pUC19-JL3 contains a 0.4 kbpEcoRI-HindIII fragment including the JL3 region from VII-L (Walton etal., 1986). This plasmid was digested with EcoRI, its ends were madeblunt, and the linearized plasmid was treated with HindIII. The JL3region was ligated into plasmid pVZ1 previously digested with HincII andHindIII. Plasmid pVZJL38 was constructed by digesting the resultingplasmid, pVZJL3, with SmaI and EcoRI; an ˜0.8 kbp EcoRI-HindIII fragmentfrom plasmid pUC19-JL8 (Walton et al., 1986), with only its HindIII endmade blunt, was ligated into the plasmid. A 1.45 kbp EcoRI fragment fromplasmid YRp7 containing the TRP1 gene (Struhl et al., 1979), was theninserted into this new plasmid, pVZJL38. The resulting plasmid,pVZJL38TRP1(+), was digested with BglII and a 3.6 kbp BamHI fragmentcontaining ADE2 was inserted (Gottschling et al., 1990). PlasmidpVZJL38TRP1(+)ADE2(−) has ADE2 inserted in the opposite transcriptionalorientation as TRP1.

YEpSIR3 (pKAN63) carries a ˜7 kbp BamHI genomic insert containing SIR3and flanking chromosomal sequences (Ivy et al., 1986), cloned into YEp13(Broach et al., 1979). CEN-SIR3 (pHR62-16) contains a 3.7 kbp HpaIfragment of plasmid pKAN63, encompassing SIR3 and its putativetranscriptional regulatory elements (Shore et al., 1984), inserted intothe SmaI restriction site of plasmid pRS314 (Sikorski and Hieter, 1989).Plasmid-23 (2m-SIR3) carries the same SIR3 fragment cloned into pHR59-33(2m), a derivative of pRS424 (Christianson et al., 1992) in which theClaI site was deleted.

Plasmid pHR49-1 was constructed by inserting a 1.2 kbp BamHI fragmentcontaining HIS3 from YDpH (Berben et al., 1991) into the BglII site ofpRS316-SIR1 (obtained from Lorraine Pillus), which contains SIR1 andflanking genomic sequences. All other plasmids used for strainconstruction have been described previously (Ivy et al., 1986; Kimmerlyand Rine, 1987; Examples I and II).

DNA manipulations were performed as previously reported (Sambrook etal., 1989; Example I). E. coli strains MC1066 (r⁻ m⁻ trpC9830 leuB600pyrF::Tn5 lacDX74 strA galU galK) (Casadaban et al., 1983), JF1754 (r⁻m⁻leuB metB hisB) (Himmelfarb et al., 1987) and TG1 (supE hsdD5thiD(lac-proAB) F′[traD36 proAB⁺ lacI^(q) lacZDM15]) (Sambrook et al.,1989) were used as plasmid hosts. Media for bacterial strains wereprepared as described (Sambrook et al., 1989). Complementation ofbacterial mutations by homologous yeast genes was used when applicable.

2. Yeast Strains and Methods

Media used for the growth of S. cerevisiae were described in Example I;all cultures were grown at 30° C. Yeast transformation was performed byelectroporation in the presence of sorbitol (Becker and Guarente, 1991)or the lithium acetate procedure (Schiestl and Gietz, 1989). 5-FOAresistance (5-FOA^(R)) was determined as described in Example II. Yeaststrains manipulations were carried out as described (Rose et al., 1990).

Strains UCC500-505 were constructed by transformation of YPH250(Sikorski and Hieter, 1989) with BamHI-digested plasmids pVURAH2(+),pVURAH2(−), pVURAH3(+), pVURAH3(−), pVURAH4(+), and pVURAH4(−),respectively. Strains UCC506-511 were constructed by transformation ofstrain YPH250 with the same plasmids digested with NotI. In both cases,Ura⁺ colonies were selected. ppr1⁻ derivatives of these strains wereconstructed by transformation with EcoRI digested pDPPR1-HIS3, andselection for His⁺ transformants.

URA3 was inserted into the ADH4 locus (about 20 kbp from telomere VII-L)of YPH250 to yield UCC1003, as described (Gottschling et al., 1990).UCC3248, UCC3249 and UCC3250 are derivatives of UCC1001 (Gottschling,1992) that are sir2::HIS3, sir3::HIS3 and sir4::HIS3, respectively, andwere created by transformation as described (Kimmerly and Rine, 1987;Example II). A sir1::HIS3 derivative of UCC1003 (UCC3243) wasconstructed by transforming UCC1003 with ClaI and SmaI digested pHR49-1.

Plasmid ph1.5HIS3(+) was digested with NotI and transformed into UCC506and UCC507 to make UCC2515 and 2517, respectively. ph1.5HIS3(−) wastransformed in the same way into UCC506 and UCC507, to generate UCC2516and UCC2518, respectively. Strains UCC2524-2527 were derived from YPH250after transformation with the various pVRUH2(+/−)HR1(+/−) constructionsdigested with SphI and NotI. UCC1005 is derived from YPH250 (Sikorskiand Hieter, 1989) by transformation with pVRURA3TEL, as described(Gottschling et al., 1990). UCC1005 was transformed with pYAHIS4-2(−)that had been digested with EcoRI and SalI, yielding strain UCC2509.Strain UCC2528 carries a telomeric URA3 at the VII-L telomere; it wascreated by transformation of YPH500 (Sikorski and Hieter, 1989) withpVII-L URA3-TEL (Example I).

The UCC2535 strain was created by transforming YPH250 withpVRUH2(−)HR1(+), selecting for His⁺ transformants, and then screeningfor Ura⁻ cells. URA3 was integrated at the ADH4 locus of UCC2535 bytransformation with padh4::URA3, as described (Gottschling et al.,1990), generating strain UCC2585. UCC2536, a meiotic segregant of across between UCC2528 and UCC2535, carries HIS3 on V-R and URA3 onVII-L. ppr1⁻ gcn4⁻ derivatives of the strains UCC2515-2518, 2524-2527,2509, 2536 and 2585 were constructed by transformation with EcoRIdigested pDPPR1::LYS2, and selection for Lys⁺ colonies; next, the GCN4locus was disrupted by transformation with pVZ1DGCN4::TRP1 digested withNotI and SalI, yielding UCC2580-2583, 2576-2579, and 2589-2591.

The gamma-deletion method (Sikorski and Hieter, 1989) was used tointroduce TRP1 and ADE2 between the JL3 and JL8 regions on VII-L (Waltonet al., 1986). Plasmid pVZJL38TRP1(+)ADE2(−) was digested with BamHI andtransformed into UCC1003 to yield strain UCC1035. The expectedstructures of the various chromosomal constructs were confirmed bySouthern analysis as described in Examples I and II. All other strainshave been described in Example I.

B. Results

1. Silencing of URA3 Decreases with Increased Distance From the Telomere

In Example I, the inventors detected telomeric position effect (TPE) inS. cerevisiae 4.9 kbp from the left end of a modified chromosome VII(VII-L) by measuring the level of transcriptional repression of atelomere-proximal URA3 when various yeast genes were inserted betweenURA3 and the telomere. However, the effect of each inserted sequence onURA3 expression was not exclusively dependent on the size of the insert.To better characterize the spread of TPE in S. cerevisiae, the inventorsexamined the expression of URA3 as a function of its distance from arepresentative telomere, without introducing any new sequences betweenURA3 and the end of the chromosome.

A set of isogenic strains was created with URA3 placed at variousdistances from the right end of chromosome V (V-R); the normalchromosomal copy of URA3 is non-functional in each strain. At each siteof insertion, URA3 was positioned in either transcriptional orientation.This set of strains may be divided into two groups: those thatmaintained the original ˜6.7 kbp telomere-associated Y′ element of V-R,and those in which the Y′ and some adjacent sequences were replaced witha new terminus of (TG₁₋₃)_(n). These Y′ elements are middle-repetitiveDNA sequences found proximal to some but not all yeast telomeres; theirfunction is unknown (Olson, 1991).

Transcriptional repression as a function of distance from the chromosomeend was analyzed by determining the level of URA3 silencing in eachstrain. The level of silencing in a population of cells is quantified bydetermining the fraction of cells capable of forming colonies on5-fluoroorotic acid (5-FOA) medium; 5-FOA is lethal to cells expressingthe URA3 gene product (Boeke et al., 1987). In the inventors' analysis,the ability of a cell to give rise to a colony on 5-FOA (5-FOA^(R))indicates that when it was plated onto the medium, the cell containedlittle or no URA3 gene product. Thus when URA3 is telomeric,telomere-mediated transcriptional repression enables the cell to grow on5-FOA (Gottschling et al., 1990).

Quantification of TPE spreading showed that, when the fraction of5-FOA^(R) cells is plotted versus the distance of the URA3 promoter fromthe telomere, a continuous gradient in frequency of silencing isobserved, with the highest frequency occurring at the mosttelomere-proximal position. Repression was no longer detected when theURA3 promoter was located 3.5 kbp away from the telomere. The steadydecrease in frequency of repression with respect to promoter distancefrom the telomere suggested that the position of the URA3 promoter wasthe key element in determining repression; transcriptional orientationwith respect to the telomere did not appear to be significant inregulating URA3 expression. Finally, in strains with a Y′ elementbetween the URA3 gene and the V-R telomere (UCC500-505), no repressionwas detected at the tested distances of 10 kbp to 16 kbp from thetelomere.

2. Absence of a Transactivator Increases the Extent of TPE Spreading

If promoter distance from the telomere is a primary determinant forgoverning TPE spreading, then weakening the promoter might result in anincrease in spreading. To test this, ppr1⁻ derivatives of the strainsdescribed above, with URA3 at various distances from the telomere, werecreated. PPR1 is a transactivator protein that enhances expression ofthe URA3 gene (Loison et al., 1980; Roy et al., 1990). Repression wasmore frequent at each location of URA3, and detectable over a greaterdistance from the telomere in ppr1⁻ than in PPR1⁺ strains. Thus therange over which TPE spreads seems to be inversely related to thepromoter strength of the gene being assayed. Similarly, deleting GCN4,the HIS3 transactivator (Hope and Struhl, 1985; Hinnebusch, 1988),reduced the ability of strains carrying a telomeric copy of HIS3 to formcolonies on medium lacking histidine, indicating that this effect is notspecific to URA3.

In the ppr1⁻ strains with the Y′ element present at V-R, a smallfraction of 5-FOA^(R) cells were reproducibly observed in the twostrains in which the URA3 promoter is about 11 and 12 kbp from thetelomere. Southern analysis revealed no change in chromosome structurebetween URA3 and the telomere in these strains. These results contrastwith the data for strains lacking the Y′ element on V-R (UCC518-523), inwhich no repression was detected beyond ˜6 kbp from the V-R telomere.Thus it seems that 6.7 kbp of Y′ sequence has a greater ability tosustain telomere-dependent silencing than the same length of unique V-Rsequence.

3. Overexpression of SIR3 Enhances TPE Spreading

The gene products of SIR2, SIR3, and SIR4 are required for TPE, and ithas been postulated that one or more of them is a structural componentof silent yeast chromatin (Nasmyth, 1982; Ivy et al., 1986; Marshall etal., 1987; Rine and Herskowitz, 1987; Alberts and Sternglanz, 1990;Johnson et al., 1990; Example II; Stone et al., 1991). To examinewhether the normal cellular level of SIR2, SIR3, or SIR4 limits therange of silent telomeric domains, the inventors tested whetherintroduction of multiple copies of the SIR2, SIR3, or SIR4 genes wouldincrease the spread of TPE. Only raising SIR3 copy number enhancedposition-effect spreading on telomere-adjacent genes. No phenotype wasobserved in strains transformed with a multicopy plasmid carrying SIR2.Increasing SIR4 dosage relieved silencing on telomeric genes; a similareffect has been previously observed at a weakened HMR (Sussel and Shore,1991).

The effect of SIR3-overexpression was quantified in the previouslydescribed sets of strains. Increased dosage of SIR3 raised the frequencyof URA3 silencing in each strain. In ppr1⁻ strains overexpressing SIR3on a high-copy plasmid (YEpSIR3), URA3 was frequently silenced 16 kbpfrom the telomere (with a Y′), while in cells with vector alone (YEp13)no significant silencing was detectable beyond 4 kbp. Similar resultswere obtained in PPR1⁺ strains transformed with YEp13 or YEpSIR3,although as expected from the data presented in the previous section,URA3 transcription was somewhat less frequently repressed than in theppr1⁻ strains. Again, the presence of a Y′ element appeared tofacilitate TPE spreading over longer distances than unique chromosomalsequences.

Extrapolation of the “YEpSIR3 with Y′” curve suggested that TPEspreading should extend inward ˜25 kbp from the end of chromosome V-R inthe SIR3-overexpressing strains. Consistent with this estimate, URA3 wasrepressed at 22 kbp from the VII-L telomere when SIR3 was overexpressed,but URA3 expression was not affected at its normal locus, ˜120 kbp fromtelomere V-L (Mortimer et al., 1992). No increase in telomeric silencingwas detected in strains transformed with plasmids carrying mutantalleles of SIR3, indicating that propagation of telomeric silencing isdependent on functional SIR3. These results are consistent with SIR3being a limiting component required to assemble repressive telomericchromatin.

If SIR3 is indeed limiting, the spread of TPE should be very sensitiveto SIR3 gene dosage. This hypothesis was tested in ppr1⁻ strainstransformed with SIR3 carried either on a centromeric (CEN-SIR3) or amulticopy plasmid (2m-SIR3), or with the vectors alone. With asingle-copy plasmid (CEN-SIR3), the spreading effect was indeed lessenhanced than with a high-copy plasmid (2m-SIR3), but greater than witheither vector alone. Hence, the results indicate that SIR3 dosage limitsthe spread of yeast telomeric position-effect.

4. Increased SIR3 Dosage Cannot Suppress the Requirements of SIR2, SIR4,NAT1, ARD1, and Histone H4 for TPE

In addition to SIR3, the gene products of SIR2, SIR4, NAT1, ARD1 andHHF2 (histone H4) are required for transcriptional silencing attelomeres (Example II). The inventors tested whether the increaseddosage of SIR3 could restore TPE in cells deficient for these otherproteins. Strains containing URA3 adjacent to the VII-L telomere anddefective in each of the aforementioned genes, were transformed with ahigh-copy SIR3 plasmid. In no case did increased levels of SIR3 restoretelomeric silencing.

Mutations in SIR1 do not relieve silencing at telomeres, suggesting thatSIR1 is not involved in controlling TPE (Example II). Consistent withthis idea, SIR3-overexpression in sir1⁻ strains enhanced TPE spreading,as observed in wild-type strains. Since the SIR3 dosage-dependentenhancement of TPE cannot suppress the requirements for SIR2, SIR4,NAT1, ARD1, and histone H4, it appears that the SIR3-effect operatesthrough the normal mechanism of telomeric silencing, rather thanintroducing a novel mechanism of silencing.

5. Silenced Chromosomal Domains Spread Continuously from the Telomere

The results presented above suggest that the silenced telomeric domainspreads inward along the chromosome in a continuous fashion. To furthertest this idea, two genes were placed adjacent to one another near thesame telomere, and the transcriptional state of the centromere-proximalgene was examined when the telomere-proximal gene was transcriptionallyactive. If the silenced domain is indeed spread continuously along thechromosome, then the centromere-proximal gene should always bederepressed when the telomere-proximal gene is active. However, if therepressed domain is discontinuous, then the centromere-proximal gene maybe in a repressed state even when the telomere-proximal gene is active.

Both the URA3 and HIS3 genes were inserted near the V-R telomere withouta Y′ element present; each of the eight possible permutations of URA3and HIS3 located near the V-R telomere was constructed. In addition,three strains were created in which URA3 and HIS3 were located on twodifferent chromosomes (V-R and VII-L), either with both genes adjacentto a telomere (UCC2590), or URA3 at a telomere and HIS3 non-telomeric(UCC2589), or the converse situation (UCC2591). In order to improve thesensitivity of the spreading assay, the promoters of URA3 and HIS3 wereweakened by deleting PPR1 and GCN4, the genes which encode theirrespective transactivators, in each strain. All strains grew in theabsence of histidine, indicating that HIS3 was capable of beingexpressed at each chromosomal position, although expression wascompromised at some telomeric locations (e.g. UCC2577, colony size wassmall and plating efficiency was reduced on “-his”). All strainscarrying a telomeric URA3 gave rise to colonies which grew onfully-supplemented 5-FOA medium, reflecting transcriptional repressionof URA3.

In the four strains with both URA3 and HIS3 located near the V-Rtelomere, and HIS3 as the telomere-proximal marker (UCC2576-2579), nogrowth was detected on “FOA-his” medium. That is, when HIS3 was nearerto the telomere and transcriptionally active, URA3 was nevertranscriptionally repressed. In contrast, when URA3 wastelomere-proximal (UCC2580-2583) colonies were obtained on FOA-his,indicating that it was possible for URA3 to be repressed while HIS3 wasactive. Thus TPE spreads continuously inward from the telomere. Theseresults also suggest that the spread of silencing can be blocked bytranscription of an intervening gene.

Of the four strains with URA3 in the telomere-proximal location, UCC2581showed conspicuously poor growth on FOA-his. In this strain, the URA3and HIS3 promoters are separated by only ˜0.5 kbp. In such closeproximity it might be difficult to open the HIS3 chromatin structurewithout also disrupting the silencing apparatus over URA3. Anothernotable result was observed when URA3 and HIS3 were located at differenttelomeres (UCC2590); robust colonies grew on “FOA-his”, indicatingrepression at one telomeric locus while the other telomeric marker wasexpressed. This result indicates that telomeric silencing islocus-specific.

The inventors then examined whether the increased spread of silencingmediated by SIR3-overexpression was also continuous. TRP1 and URA3 wereinserted ˜12.5 and 22 kbp, respectively, from the VII-L telomere.5-FOA^(R) colonies were observed only when the cells were transformedwith YEpSIR3; however, no 5-FOA^(R) was detected if TRP1 wassimultaneously expressed in these cells. TRP1 expression by itself wasonly modestly impaired in YEpSIR3-transformants, as demonstrated bytheir high efficiency of plating on “-trp-leu”. Similar results wereobtained when ADE2 (inserted ˜9 kbp from the same telomere) replacedTRP1 in this study. Taken together, these observations suggest that SIR3propagates silencing continuously from the telomere.

C. Discussion

The inventors have carried out a systematic characterization of thespreading of telomeric position effect (TPE) in Saccharomycescerevisiae. The telomeric position effect in yeast can be considered asa gradient of transcriptional silencing along the chromosome. Theinventors postulate that this gradient reflects the limited assembly ofa silent chromatin (heterochromatic-like) structure that initiates atthe telomere and proceeds continuously inward along the chromosome. Inthe inventors' analysis, the fraction of 5-FOA^(R) cells provided anestimate of the frequency at which a telomeric URA3 was located withinthis repressive structure.

Transcriptional inactivation of a telomeric locus may be viewed as thefinal product of a reaction in which subunits of silent chromatin areassembled. In a simple model, silencing of a URA3 gene six kbp from thetelomere would require six times as many subunits than that needed tosilence a URA3 gene located one kbp away. If the assembly of telomericrepressive chromatin were a first-order reaction, then the occurrence ofa repressed URA3 gene at one kbp from the telomere would be expected sixtimes as frequently as when URA3 is six kbp away. This Example showsthat this is not the case. An exponential function more aptly describesthe relationship between frequency of silencing and distance from thetelomere. Rather the data suggest that telomeric silencing results fromthe cooperative assembly of subunits, and/or assembly of multiplecomponents. A multimeric representation of silent chromatin is expectedto involve the four core histones plus additional components(Eissenberg, 1989; Henikoff, 1990; Spradling and Karpen, 1990;Grigliatti, 1991), as quantitated in vivo in this Example.

It has been proposed that specific terminator sequences along thechromosome act as barriers to heterochromatic spreading (Tartof et al.,1984). No such regions were detected on the telomere-proximal 16 kbp ofV-R, nor over 20 kbp of a modified VII-L, although these data do notrule out the existence of such sites in yeast.

Cells carrying URA3 and HIS3 located near the V-R telomere, with HIS3telomere-proximal, were unable to form colonies on FOA-his media. Sincethis medium selects for cells in which both URA3 is repressed and HIS3is active, this result demonstrates that silent telomeric domains arecontinuously propagated from the end of the chromosome in yeast. Since atelomeric gene can be induced to become active Example I, the inventorssuggest that transcription may actively block silent chromatinpropagation. Alternatively, transcription may not act as a barrier tothe spread of silencing per se, but rather reflect that the silenttelomeric domain assembled only a short distance from the telomere, thusnever encompassing the HIS3 (or URA3) gene. The distinction betweenthese two models should be considered in thinking about gene regulationwithin chromosomal domains.

1. The Role of the Promoter in TPE Spreading

The presence of silent chromatin structures over a telomeric locusappears to impede the access of sequence-specific DNA-binding proteinsto the DNA within, thereby generating a TPE (Examples I and II;Gottschling, 1992). These data show a steady decrease in the frequencyof silencing compared to the distance of the URA3 promoter from thetelomere. This result strongly suggests that a gene's promoter is amajor determinant in cis for effective transcriptional repression neartelomeres. Combined with the finding that silencing of URA3 does notappear to be dependent on the transcriptional orientation of URA3, theinventors propose that repression is primarily exerted on the gene'spromoter, and therefore blocks initiation rather than elongation.

Two important points about position effect are provided by the studiesin which PPR1 was deleted. As with most transactivator proteins, PPR1appears to modulate transcription through the promoter (Roy et al.,1990). Hence, the increased frequency of telomeric silencing of URA3 inppr1⁻ strains supports the result that promoter occlusion is critical inachieving position effect repression. These results also suggest thatspreading of position effect is a function of promoter strength of thegene being assayed.

A position effect on timing of replication has been detected at ˜35 kbpfrom the V-R telomere (Ferguson et al., 1991; Ferguson and Fangman,1992), while position effect on URA3 transcription is not detectedbeyond ˜13 kbp from the same terminus. At present the inventors cannotresolve whether this apparent discrepancy reflects differences betweenthe two assays being used, or inherent distinctions between themechanisms of initiating replication and transcription.

2. Effect of Y′ Elements on the Spread of Telomeric Silencing

It has been suggested that Y′ elements overcome telomere position effect(Greider, 1992), since genes embedded into Y's are not transcriptionallyrepressed (Carlson et al., 1985; Louis and Haber, 1990). However, thesedata argue that Y's do not block the spread of telomeric repression perse; the inventors find that a 6.7 kbp Y′ element sustains a greaterfrequency of silencing than an equal length of unique chromosomalsequences. It is unclear whether Y's are involved in propagation orreinitiation of silencing, or if Y's simply lack elements present inunique chromosomal DNA which suppress the spreading oftelomere-dependent transcriptional inactivation. Nevertheless, thepresence of a Y′ element adjacent to a telomere results in a moreextensive silent chromosomal domain. Perhaps this trait is important inmaintaining the unique telomeric presence of Y′ elements.

3. SIR3 Enhances Position Effect in Yeast

Overexpression of SIR3 enhances position-effect variegation of telomericgenes; this SIR3-effect was also detected within and adjacent to the HMloci. Thus the modulation by SIR3 of position-effect repression islikely to occur at other places in the genome where an initiation sitefor SIR3-dependent silencing resides.

The slope of the observed gradient in frequency of URA3 silencing alongV-R is altered by overexpressing SIR3 in the cell, suggesting that, incontrast to the effect of a ppr1 mutation, SIR3-overexpression affectssilent chromatin rather than an intrinsic property of URA3. In addition,the increase in telomeric silencing is sensitive to SIR3 gene dosage,indicating that SIR3 is limiting in the cell. These data suggest thatSIR3 may be a structural component of yeast repressive chromatin, or afactor directly required for its assembly. Alternatively, SIR3 may actindirectly by regulating the level or activity of structural or assemblyconstituents of silent chromosomal domains.

SIR3 bears no significant similarity to any known enhancers of positioneffects, such as the Drosophila Su(var)2-5 (HP-1) or Su(var)3-7 proteins(Alberts and Sternglanz, 1990), nor does it harbor a detectablechromodomain motif, which is thought to mediate the packaging ofheterochromatin by the Su(var) 2-5 and Polycomb gene products (Paro andHogness, 1991; Messmer et al., 1992). Extragenic suppressor analysis ofHML silencing indicates a physical interaction between SIR3 and histoneH4 (Johnson et al., 1990). Thus the inventors favor the model that SIR3directly interacts with yeast nucleosomes to facilitate the compactionof chromatin into a higher-order structure responsible for silencedregions of the yeast genome. In this light, SIR3 may be a functionalequivalent of histone H1, mediating supranucleosomal organization of thegenome (Weintraub, 1984).

In addition to histone H4, telomeric silencing requires the products ofSIR2, SIR4, NAT1 and ARD1. The roles of SIR2 and SIR4 in transcriptionalrepression are not yet clear. NAT1 and ARD1, which are subunits of anN-terminal acetyltransferase (Park and Szostak, 1992), presumably modifychromatin component(s) to facilitate assembly of repressed chromosomalstates (Mullen et al., 1989; Park et al., 1992).

The ability of telomeric silencing to spread along the chromosome raisesthe question as to whether a cell can control the size of silenceddomains. This issue is particularly critical for S. cerevisiae, in whichinappropriate regional silencing might have immediate deleteriouseffects, due to the high density of genes along the chromosome (Olson,1991). A cis-element can act as a chromosome-specific barrier againstthe spread of silent domains [e.g. active transcription units (thiswork), or homologues of the Drosophila scs sequences (Kellum and Schedl,1992)]. On a cellular scale, limiting the amount of SIR3 in the cellcould prevent excessive transcriptional inactivation of the entiregenome. Since the SIR3 gene is itself located near a telomere (Ivy etal., 1985), and no essential gene has been found between SIR3 and thetelomere (Basson et al., 1987; Brisco et al., 1987; Dietzel and Kurjan,1987; Mortimer et al., 1992), position-effect repression of the SIR3locus would provide a plausible negative feedback mechanism for controlof position-effect spreading in yeast. If telomeric chromatin spread asfar as the SIR3 locus, transcription of SIR3 would be repressed, thuslimiting further spreading of the repressive chromatin. In apparentcontrast to the yeast genome, larger eukaryotic genomes are extensivelyheterochromatic. This may be due to the presence of more abundantfunctional homologue(s) of SIR3. Extensive but carefully controlledheterochromatization of chromosomes may play a major role in control ofcellular differentiation and development in complex eukaryotes.

This Example shows that the spread of telomeric position effect in S.cerevisiae is modulated by numerous factors, including promoter distancefrom the telomere, promoter strength, transcriptional status oftelomere-proximal genes, presence of Y′ elements, and intracellularconcentration of the SIR3 gene product.

EXAMPLE IV A Transactivator Competes to Establish Gene Expression in aCell Cycle Dependent Way

In multicellular eukaryotes, chromosomal position effects generallyinvolve the repression of a euchromatic, wild-type gene when it has beenplaced in or near heterochromatin as the result of a chromosomalrearrangement (Lima-de-Faria, 1983). In a population of cells with sucha rearrangement, the gene may escape repression; consequently, theresulting phenotype is variegated, exhibiting patches of normal andmutant tissue. A classic example of this phenomenon is the mosaicred-and-white eye of Drosophila in which the white gene has beentranslocated within centromeric heterochromatin (Eissenberg, 1989;Henikoff, 1990; Spradling and Karpen, 1990).

When a wild-type gene is located near a telomere in the budding yeastSaccharomyces cerevisiae, it too is subject to position-effectvariegation (Example I). For instance, when yeast cells with the ADE2gene placed near a telomere form a colony on solid medium, the colony iscomposed of sub-populations in which the ADE2 gene is either expressed(white sectors) or repressed (red sectors). The different phenotypes ofthe sectors in a colony reflect the ability of genetically identicalcells to switch between phenotypic states. The fact that large sectorsare phenotypically uniform reflects the ability of each state to beheritably propagated for multiple generations.

Similarly, yeast cells with a telomeric URA3 gene can form colonies onmedium containing 5-FOA, a drug lethal to cells expressing URA3 (Boekeet al., 1987), indicating that the cells are phenotypically ura3⁻.However, these 5-FOA resistant cells can form colonies when placed onmedium lacking uracil, thus the cells are able to switch theirphenotypic status and induce expression of the telomeric URA3 gene(Example I).

Silencing of telomeric genes in S. cerevisiae is likely due to astructurally distinct chromatin domain that initiates at the telomere.Evidence for this specialized chromatin structure includes:identification of mutations in the histone H3 and H4 genes which relievetelomeric silencing (Example II) the finding that telomere-adjacentchromatin contains histone H4 in a hypoacetylated state compared to H4in actively transcribed chromatin regions of the genome (Braunstein etal., 1993), and the relative inaccessibility of telomere-proximal DNA toin vivo modification by the E. coli dam methyltransferase protein(Gottschling, 1992).

In addition, the frequency with which a gene is silenced decreases withincreasing distance from the telomere, suggesting that the structurenucleates at the telomere and the extent of its inward assembly alongthe chromosome varies between cells (Example III; Renauld et al., 1993).The extent of this assembly is proportional to the cellularconcentration of SIR3, a gene product required for silencing attelomeres and the silent mating loci, HML and HMR (Example II; Laurensonand Rine, 1992; Example III). These results suggest that SIR3 israte-limiting for assembly of the silent chromatin structure, andimplicate SIR3 as a component of the silent structure.

Questions that arise in the study of position effect variegation are howdoes a gene switch between phenotypic states and, once a state isdetermined, how is it heritably propagated (Brown, 1984; Weintraub,1985). With respect to position-effect variegation and the firstquestion, two models of regulation that involve a role for chromatinstructure have evolved (Felsenfeld, 1992). Both models propose thattranscription of a gene is inhibited by assembly of its DNA intochromatin. Furthermore, one or more transcriptional activator proteins(transactivators) bind in a sequence-specific manner to DNA located inproximity to the gene and facilitate transcription of that gene, thusovercoming the chromatin's repressive nature. Where the models differ isthat in one case chromatin prevents the transactivator from gainingaccess to the DNA, in essence keeping the gene ‘irreversibly’ repressed.However, during DNA replication the chromatin structure of the gene isperturbed and the transactivator has the opportunity to gain access andestablish transcription, before re-assembly of the chromatin iscompleted. In the second case, the transactivator can induce genetranscription at anytime in a replication-independent manner,effectively disrupting the repressive nature of the chromatin.

At its normal locus, URA3, like many biosynthetic pathway genes, isconstitutively expressed at a basal level, but can be induced to higherlevels of expression (Lacroute, 1968). URA3 induction is contingent uponbinding of an activated form of the transactivator PPR1 to the UpstreamActivating Sequence (UAS) of the gene (Losson and Lacroute, 1981; Roy etal., 1990). Interestingly, when URA3 is located adjacent to a telomereits basal level of expression may be repressed, since the cells arephenotypically ura3⁻ (Example I).

This Example concerns how a gene located near a telomere overcomessilencing. Specifically, the inventors examined the role of PPR1 in theexpression of a telomeric URA3 gene. The results show that silenttelomeric chromatin inhibits basal expression of URA3 and prevents thetranscriptional activation by PPR1 of the telomeric URA3 gene in G₁ andearly S phases of the cell cycle, in addition to when cells are arrestedin G₀. Furthermore, this suggests that upon replication of the telomericDNA, a competition takes place between assembly of a silent chromatinstructure and assembly of a PPR1-mediated transcriptionally active gene.

A. Methods

1. Plasmid Constructions

Plasmid FAT-PPR1 was constructed by ligating a 4.4 kbp EcoRI fragmentcontaining the PPR1 gene (from pUC8-PPR1, obtained from R. Losson) intoplasmid YEpFAT10 (referred to as “FAT”; 2μ ARS, TRP1, leu2-d, obtainedfrom K. Runge; Runge and Zakian, 1989). A 3.7 kbp HindIII-SphI fragmentcontaining the entire PPR1-1 allele (from plasmid pFL11; Losson andLacroute, 1983) was inserted into plasmid pVZ1 (Henikoff andEghtedarzadeh, 1987). The resulting plasmid (pVZPPR1-1) provided a 3.7kbp HindIII-BamHI fragment containing PPR1-1 which was ligated intopRS425 (Sikorski and Hieter, 1989) to yield plasmid pRS4-PPR1-1.

Plasmids pRS305-GALPPR1-1 and pRS305-GALppr1-1 were constructed in aseries of steps. A 685 bp EcoRI-BamHI fragment containing the GAL1,10promoter (Johnston and Davis, 1984, from pBM150) was ligated intoEcoRI-BamHI digested pRS314 (Sikorski and Hieter, 1989), the resultingplasmid (pRS314GAL) was digested with ApaI-EcoRI and a 2.8 kbpApaI-EcoRI fragment containing the 3′ portion of PPR1-1 from plasmidpRS4-PPR1-1 was inserted yielding pRS3GAL3′PPR1-1. Next, a 500 bpfragment containing the 5′ portion of the PPR1-1 allele was produced byPCR amplification (Innis et al., 1990). The primers were designed tointroduce an EcoRI site 28 bp upstream of the PPR1 ATG initiation codonand to include the EcoRI site within the PPR1-1 coding sequence(PPR1-ATG oligo, 5′-CCGGAATTCATACGAAGATGATGATTAAATC-3′, SEQ ID NO:6, thenew EcoRI site is underlined; PPR1-n650 oligo,5′-GGCTTGCCATAGACTTGCTCG-3′, SEQ ID NO:7). The fragment was digestedwith EcoRI and inserted between the GAL1,10 promoter and the 3′ PPR1-1sequence in pRS3GAL3′PPR1-1; one orientation of the insert yieldedpGALPPR1-1 which has the GAL1,10 promoter fused to the entire PPR1-1coding sequence (GALPPR1-1), while the other orientation of the insertyielded pGALppr1-1 which has the 5′ portion of the PPR1-1 alleleinverted resulting in a mutated gene fusion (GALppr1-1). The 3.5 kbpApaI-BamHI fragments containing GALPPR1-1 and GALppr1-1 from PGALPPR1-1and pGALppr1-1 respectively, were inserted into pRS305 (Sikorski andHieter, 1989) yielding pRS305-GALPPR1-1 and pRS305-GALppr1-1.

Plasmid pVZADH4 contains the ADH4 locus on a 3.1 kbp EcoRI-SalI fragment(Example I). A 4.8 kbp HindIII-XbaI fragment containing the LYS2 genefrom plasmid pDP6 (Fleig et al., 1986) was inserted into XbaI-HindIIIdigested pVZADH4 creating pVZadh4::LYS2. The UASGAL-URA3 allele wasproduced by sequential PCR amplification steps (Ausubel et al., 1989).The primers were designed to replace the PPR1 binding site (UAS_(URA),5′-TTCGGTAATCTCCGAA-3′, SEQ ID NO:8 (Roy et al., 1990)) with a GAL4binding site (URA3-GAL-5′ oligo,5′-CGGACGACTGTCGTCCGTCAAAAAAATTTCAAGGAAACCG, SEQ ID NO:9, URA3-GAL-3′oligo, 5′-CGGACGACAGTCGTCCGCAGAAGGAAGAACGAAGGAA, SEQ ID NO:10, the GAL4binding sequence is underlined (Verdier, 1990)).

The UAS_(GAL)-URA3 PCR product was digested with SalI and BamHI andinserted into pRS315(-PstI) producing pRS315(-PstI)-GALURA3; the PstIsite in pRS315 was previously deleted by digestion of pRS315 (Sikorskiand Hieter, 1989) with PstI, making the ends blunt with T4 DNApolymerase, and religating the plasmid. Plasmid pRS315(-PstI)-GALURA3was digested with HindIII and SmaI and religated, resulting in theUAS_(GAL)-URA3 fragment being inverted in the vector to yieldpRS315(-PstI)-GALURA3-flip.

This plasmid provided a 1.1 kbp HindIII-BamHI fragment containingUAS_(GAL)-URA3 which was inserted into pVII-L URA3-TEL (Example I) toproduce pADH4GALURA3TEL; the same 1.1 kbp HindIII-BamHI UAS_(GAL)-URA3fragment was inserted into HindIII-BamHI digested pVZADH4 resulting inplasmid pΔadh4::GALURA3. A 1.2 kbp HindIII-NotI fragment (madeblunt-ended with T4 DNA polymerase, from pVII-L URA3-TEL) was ligatedinto HindIII (made blunt ended with T4 DNA polymerase) digestedpVZadh4::LYS2 producing pURA3-TEL-LYS2. A 1.5 kbp PstI fragment (frompADH4GALURA3TEL) containing the UAS_(GAL)-URA3 promoter was insertedinto PstI digested pURA3-TEL-LYS2 to replace the wild-type URA3promoter; the resulting plasmid was pGALURA3-TEL-LYS2.

A 1.35 kbp BamHI fragment containing the entire URA1 gene (Roy, 1992)produced by PCR amplification of genomic DNA (5′URA1 oligo,5′-CGAACGGATCCCCTTCAGCCACTACAGCCTACTT-3′, SEQ ID NO:11; 3′URA1 oligo,5′-CGAAGGGATCCGCCAATTGCGAATGCACTCACCG-3′, SEQ ID NO:12, the BamHI sitesare underlined) was inserted into pVZ1 to yield plasmid pVZURA1. A 1.1kbp HindIII-BamHI URA3 fragment was ligated into HindIII-BamHI digestedplasmid YDpK (Berben et al., 1991), yielding plasmid YDpK-URA3. Plasmidp5′URA3 contains a 415 bp HindIII-EcoRV 5′ URA3 fragment ligated intoHindIII-EcoRV digested pVZ1. Plasmid CY807+TRP1 (bar1::TRP1) wasconstructed by inserting a 723 bp BamHI fragment containing TRP1, fromYDp-W (Berben et al., 1991), into the BglII site in the BAR1 sequence inplasmid CY807 (obtained from S. Honigberg).

Plasmids pBM292 (GAL4-wild-type, 881 amino acids), pBM430 (GAL4, C-term.amino acid 292), pBM433 (GAL4, C-term. amino acid 684), pBM789 (GAL4,C-term. amino acid 174), and pBM1268 (GAL4, C-term. amino acid 383) areCEN, TRP1 plasmids, as described by Johnston (1988). Plasmids pBD57 andpJM206 were obtained from F. Cross, and plasmid pPL9 was obtained fromR. Surosky (1992).

2. Yeast Methods and Strains

S. cerevisiae were grown at 30° C.; liquid cultures were agitated duringincubation at 180 RPM. All studies in liquid culture were carried outwith mid-log phase cells unless otherwise indicated. Plating efficiencyanalysis and synthetic media have been described previously (Example I),except for α-aminoadipate containing medium which was prepared asdescribed in (Sikorski and Boeke, 1991). Studies involving galactosecontrol employed YEP-3% raffinose, and 0.3% galactose for inductionunless otherwise indicated.

For studies involving drug or α-factor washout, cells were pelleted bycentrifugation for three minutes at 1500×g and washed and/or resuspendedin prewarmed medium (30°). Cells were arrested with 20 nM α-factor forthree hours, and 50 mM pthalic acid (pH=5.5) was included in the medium.For release from α-factor arrest, 1 mg/ml pronase E was included in thefresh resuspension media, except for one study where one water wash ofthe pellet was carried out and pronase E was not included in theresuspension medium. Cells were arrested with 10 μg/ml nocodazole (froma 1000× stock solution in DMSO) for three hours. Hydroxyurea wasdissolved directly in medium immediately before use to a finalconcentration of 400 mM, except in one study where it was dissolveddirectly in the cultures. Cells were fixed and stored in 10 mM Tris, 100mM EDTA, pH=8.0, 3.7% formaldehyde, and sonicated before microscopy toassess cell morphology.

S. cerevisiae were transformed using the lithium acetate procedure (Itoet al., 1983). The URA3 gene was placed adjacent to the telomeresequence (TG₁₋₃)_(n) on the left end of chromosome VII (UCC2013), orinserted at the ADH4 locus about 20 kbp from the telomere on VII-L(UCC432), as described in Example I. UCC2013 was derived from YPH499,UCC432 was derived from UCC431.

Strains UCC111, UCC113, UCC115, UCC412, and UCC2014 were constructed bytransformation of strains UCC1001, UCC1003, YPH250, UCC411, and UCC2013respectively, with plasmid pΔAPPR1::HIS3 and selection for HIS⁺transformants; this plasmid was described in Example III. StrainsUCC116, UCC117, and UCC151 were derived from strains UCC1001, UCC1003,and YPH250 respectively, by transformation with plasmid pFAT-PPR1 andselection for TRP⁺ cells; strains UCC238, UCC152, and UCC153 werederived from strains UCC1001, UCC1003, and YPH250 respectively, bytransformation with plasmid YEpFAT10 (FAT) and selection for TRP⁺.

Strain UCC411 was derived from YPH499 by transformation with HpaIdigested YDpK-URA3 and selection for LYS⁺ cells. UCC413 and UCC2016 werederived from UCC412 and UCC2014 respectively, by transformation withplasmid CY807+TRP1 digested with ClaI. Strain UCC431 was a 5-FOA^(R)(ura⁻, lys⁻) derivative of UCC413. Strains UCC409, UCC433, and UCC435were derived from strains UCC2016, UCC431, and UCC432 respectively, bytransformation with HpaI digested pRS305-GALPPR1-1; strains UCC410,UCC434, and UCC436 were derived from strains UCC2016, UCC431, and UCC432respectively, by transformation with HpaI digested pRS305-GALppr1-1.

In order to place UAS_(GAL)-URA3 (or another non-selectable marker)adjacent to telomere VII-L, a method was developed based on thephenomenon of new telomere formation at internal telomeric sequences(Example I). Plasmid pGALURA3-TEL-LYS2 was used to integrate within theADH4 locus: UAS_(GAL)-URA3 adjacent to 81 bp of telomere repeat sequencefollowed by LYS2 as the selectable marker (centromere-proximal tocentromere-distal). At a frequency of ˜10⁻⁶, loss of chromosomalsequences distal to the 81 bp internal telomeric sequence (includingLYS2) resulted in formation of a new and stable telomere having theUAS_(GAL)-URA3 gene adjacent to it.

Cells that were transformed with pGALURA3-TEL-LYS2, and were LYS⁺ andhad the correct sequences inserted within the ADH4 locus (verified byDNA blot hybridization analysis), were grown non-selectively for about25 generations. Cells which had lost LYS2 were selected for survival onmedium containing α-aminoadipate; the expected structure of telomereVII-L in the resulting lys⁻ strain was verified by DNA hybridizationanalysis.

UCC418 was derived from YM725 by transformation with NotI-SalI digestedplasmid pGALURA3-TEL-LYS2 and selection for LYS⁺ transformants; UCC420was an α-aminoadipate resistant (lys⁻) derivative of UCC418 which hasUAS_(GAL)-URA3 adjacent to telomere VII-L. UCC419 was derived from YM725by transformation with EcoRI-SalI digested plasmid pDadh4::GALURA3 andselection for URA⁺ transformants. Strains UCC419 and UCC420 weretransformed with plasmids pBM292, pBM430, pBM433, pBM789, and pBM1268,to yield strains UCC421-UCC425 respectively, for the UCC419 parent, andstrains UCC426-UCC430 respectively for the UCC420 parent. The expectedstructures of the various chromosomal constructs were confirmed by DNAblot hybridization analysis.

3. Analysis of Nucleic Acids

RNA was isolated from mid-log phase cells, unless otherwise indicated,as described in Example I. RNA hybridization analyses were performed asdescribed in Example I, except that 15 or 20 μg of total RNA wasdenatured in the presence of 20 μg/ml ethidium bromide and separated byelectrophoresis on a 1.2% agarose-5% formaldehyde (37% stock)-MOPS gel.Immediately following electrophoresis the gel was photographed andwashed twice for 15 minutes in H₂O, 15 minutes in 10×SSC and transferredto nylon (MSI, Westboro, Mass.). Photography of the gel followingtransfer verified that complete transfer of the rRNA had occurred.

RNA was immobilized on the nylon membrane by UV irradiation (120 mJ) ofthe damp membrane, followed by prehybridization of the membrane.Prehybridization and hybridization solutions contained 5×SSC, 50%formamide, 5× Denhardt's solution, 0.2 mg/ml denatured and degradedherring sperm DNA, 0.2% SDS; hybridization solution also contained 10%dextran sulfate and was filtered through a 45 μm membrane to removeparticulates. Prehybridization (1-6 hr) and hybridization (18-30 hr)were carried out at 42° C. for DNA probes and 53° C. for RNA probes.

Blots were washed five minutes at 23° C. in 2×SSC, 0.1% SDS, followed bytwo 15 minutes washes at 55° C. in 0.1×SSC, 0.1% SDS for DNA probes, orthree 20 minute washes at 60° C. in 0.1×SSC, 0.1% SDS for RNA probes,and exposed to film. The relative levels of URA3 and URA1 RNAs werequantified on a Radioanalytic Imaging System (Ambis, San Diego, Calif.).For rehybridization studies, probes were removed from the blots withthree 20 minute washes with boiling 0.2% SDS.

RNA antisense probes were labeled with ³²P-CTP or ³²P-UTP (3000 Ci/mmol)by in vitro transcription of linearized plasmids with T7 RNA polymeraseor SP6 RNA polymerase (Sambrook et al., 1989). DNA probes were labeledwith ³²P-dCTP (3000 Ci/mmol) by random oligonucleotide priming asdescribed (Sambrook et al., 1989). Plasmid p5′URA3 (T7) was the templatefor the URA3 RNA probe. Plasmid pPL9 (SP6) was the template for the ACT1RNA probe. The URA3 DNA probe was a 1.1 kbp HindIII fragment containingthe entire coding sequence, the URA1 probe was a 1.3 kbp BamHI fragmentcontaining the entire URA1 gene in plasmid pVZURA1, the SWI5 probe was a3.3 kbp HindIII fragment from pBD57, and the CLN2 probe was a 640 bpHindIII-SpeI fragment in pJM206.

B. Results

1. The URA3 Transactivator, PPR1, Is Required for Overcoming TelomericSilencing of URA3

In order to test the idea that the transactivator, PPR1, plays a role inovercoming silencing of a telomere-linked URA3 gene, the PPR1 gene wasdeleted from a strain in which URA3 was located adjacent to telomereVII-L (UCC1001). To determine whether deletion of PPR1 had a specificeffect on URA3 expression at a telomere, PPR1 was also deleted in astrain with URA3 inserted at an internal chromosomal position, the ADH4locus which is about 20 kbp from telomere VII-L (UCC1003). PPR1 was alsodeleted in a strain lacking URA3 (ura3-52; YPH250).

URA3 expression was measured by two methods: plating viability assays onmedium containing 5-fluoro-orotic acid (5-FOA) and on medium lackinguracil (-URA), and RNA blot hybridization analysis. 5-FOA is convertedinto a toxic metabolite by the URA3 gene product, such that cellsexpressing normal levels of the URA3 gene product are sensitive to5-FOA, while cells that lack it are resistant to 5-FOA (Boeke et al.,1984).

For the RNA analysis, transcript levels were analyzed from URA3, URA1,and ura3-52 (Rose and Winston, 1984, in this allele the URA3 transcriptis truncated) each of which is regulated by the PPR1 protein (Losson andLacroute, 1981). Thus, URA1 and ura3-52 RNA levels reflect the in vivolevel of PPR1 activity as a transcriptional activator in eachexperimental sample.

PPR1 was found to be required for overcoming silencing of the telomericURA3 gene. Wild type (PPR1⁺) cells with URA3 near a telomere, formedcolonies on 5-FOA medium and medium lacking uracil. This reflects theability of the telomeric URA3 gene to switch between transcriptionallyrepressed and active states. Deletion of PPR1 abolished the ability ofcells with a telomeric URA3 gene to grow in the absence of uracil.Deletion of the PPR1 binding site within the URA3 gene promoter had thesame effect as deletion of PPR1, indicating that specific binding ofPPR1 at the URA3 UAS was required for overcoming silencing. Thus, inthis telomeric context, PPR1 is required for the transcriptionalactivation of the URA3 gene.

The very small colonies which arose on -URA medium from the ppr1⁻ strainwith a telomeric URA3 gene had acquired trans-acting mutations or localchromosomal rearrangements which permitted expression of URA3.Therefore, essentially no URA3 gene product was produced from thistelomeric site when PPR1 was absent from the cell. In contrast, deletionof PPR1 had no effect on 5-FOA or -URA viability when URA3 was locatedat an internal chromosomal locus. This result suggests that at aninternal location transcription of URA3 still occurs, independently ofPPR1, and is consistent with URA3 regulation at its normal chromosomallocus (Losson et al., 1985). As expected, PPR1 deletion had no effect onthe plating viability of cells lacking a functional URA3 gene.

Telomeric URA3 mRNA was undetectable when PPR1 was deleted. However,PPR1⁺ cells with a telomeric URA3 maintained the ability to activateURA3 transcription. Deletion of PPR1 had little or no effect onexpression of an internal copy of URA3, or on expression of URA1.

Both the plating viability on -URA medium and the RNA analysis indicatethat the constitutive or basal (PPR1-independent) expression of URA3 attelomere VII-L is repressed by the telomeric silencing machinery.However, the transactivator, PPR1, is able to circumvent the telomericrepression, thus facilitating URA3 expression.

2. Increased PPR1 Dosage Prevents Silencing of a Telomeric URA3

Since a telomeric URA3 could exist in either an active or repressedstate, and because PPR1 was required for the active state, the inventorspostulated that PPR1 might compete against the assembly of a repressedstate. If this hypothesis were true, then increasing the dosage of PPR1should increase the frequency with which an active state is established.

To test this hypothesis, PPR1 was expressed from a multi-copy plasmid(FAT-PPR1, FAT is the vector alone) in strains with URA3 absent, URA3 ata telomeric, or URA3 at an internal chromosomal locus. Cell viability ofthe resulting strains was quantified on 5-FOA medium and medium lackinguracil. Increase of PPR1 protein concentration from FAT-PPR1 (verifiedby ura3-52 and URA1 RNA levels and quantitative electrophoretic mobilityshift analyses) resulted in complete 5-FOA-sensitivity of cells withURA3 at the telomeric locus, along with improved growth on -URA. Asexpected, viability was not affected by overproduction of PPR1 when URA3was at the internal locus or absent. Thus, high levels of PPR1 competeagainst telomeric silencing to perpetually maintain the URA3 gene in anactive state. These results also suggest that in a wild type cell, theconcentration of PPR1 is limiting for telomeric URA3 expression.

3. GAL4 Can Overcome Telomeric Silencing

To determine if the ability of PPR1 to overcome telomeric silencing onURA3 transcription was a general characteristic of transcriptionalactivator proteins, the PPR1 binding site upstream of the URA3 gene wasreplaced with a binding site for the GAL4 transactivator protein(Verdier, 1990). This modified URA3 gene (UAS_(GAL)-URA3) was placednext to telomere VII-L (UCC420) or within the ADH4 locus (UCC419) instrain YM725 (gal4⁻, gal80⁻, ura3⁻). The gal80 mutation relievesnegative regulation of the GAL4 protein so that activity of GAL4 isproportional to its concentration (Johnston, 1987). UAS_(GAL)-URA3 wassilenced when placed at telomere VII-L, as the cells were5-FOA-resistant and Ura⁻, but UAS_(GAL)-URA3 was not repressed wheninternally located on the chromosome since cells were 5-FOA-sensitiveand URA⁺.

The wild-type GAL4 protein or a series of C-terminal truncations of theGAL4 protein were expressed in the strains with UAS_(GAL)-URA3 locatedat the telomere or at the internal locus. The C-terminal truncationderivatives of GAL4 maintain the N-terminal DNA binding domain and bindto UAS_(GAL) in vitro, but are defective in transcriptional activationin vivo (Johnston and Dover, 1988). Expression of wild-type GAL4, from asingle copy centromeric plasmid, completely reversed silencing of thetelomeric UAS_(GAL)-URA3, as indicated by the sensitivity of this strainto 5-FOA, and robust growth on -URA medium. None of the truncated GAL4derivatives were able to activate UAS_(GAL)-URA3 adjacent to thetelomere. Expression of GAL4 or its derivatives had no effect on5-FOA-sensitivity, or -URA viability, of strains with UAS_(GAL)-URA3located internal on the chromosome. It appears that the activationdomain of GAL4 is required to compete for telomeric gene expression.These results suggest that the ability to overcome telomeric silencingis a general function of transactivators.

4. Modulating the Dosage of PPR1C Reveals that its Accessibility to theTelomeric URA3 Gene is Limited

The finding that PPR1 dosage has a demonstrable effect on telomeric URA3expression, but not for internal URA3 expression, suggested that thetelomeric URA3 gene is relatively resistant to transcriptionalactivation by PPR1 compared to when URA3 gene is locatednon-telomerically.

To investigate this, a chimeric gene, GALPPR1-1, was constructed withthe coding sequence of the PPR1-1 allele under control of the GAL1,10promoter (Johnston and Davis, 1984). The PPR1-1 allele encodes aconstitutively active protein, PPR1^(c); thus, the level of PPR1^(c)activity as a transactivator is directly proportional to its totalcellular concentration (Losson and Lacroute, 1983). The GAL1,10 promoterpermitted precise regulation of PPR1^(c) protein concentration withinthe cell (Durrin et al., 1991), since the intracellular level ofPPR1^(c) was proportional to the level of galactose in the medium (basedon ura3-52 RNA levels and quantitative electrophoretic mobility-shiftanalyses). As a control, a non-functional version of the gene fusion(GALppr1-1), which contains an inversion within the PPR1-1 codingsequence, was also created. These gene fusions were inserted at the leu2locus in isogenic ppr1⁻ strains containing URA3 at a telomeric (UCC2016)or internal chromosomal locus (UCC431) or in which URA3 was absent(UCC432).

The resulting strains were tested for viability on 5-FOA and -URA mediumthat also contain galactose. Expression of the GALPPR1-1 fusion, but notthe mutated GALppr1-1 fusion, effectively overcame silencing of thetelomeric URA3 in all cells of the population; the cells were URA⁺ and5-FOA-sensitive. Expression of GALPPR1-1 or GALppr1-1 had no effect onthe 5-FOA sensitivity or the -URA viability of strains with URA3 at theinternal locus or absent.

Levels of mRNA were analyzed from these strains grown in rich mediumcontaining 3% raffinose and 0.25% galactose, which induced expression ofGALPPR1-1 or GALppr1-1. Expression of GALPPR1-1 strongly activatedtranscription from URA3, URA1, and ura3-52, although compared toexpression of the internal URA3 gene, expression of the telomeric URA3was reduced. Equivalent levels of PPR1^(c) activity (based on URA1 andura3-52 mRNA levels, and electrophoretic mobility-shift analyses) werepresent in the GALPPR1-1 strains. This result supports the idea that,compared to the internal URA3, the telomeric URA3 gene is relativelyresistant to transcriptional activation at this concentration ofPPR1^(c).

The inventors compared the relative expression levels of the telomericURA3 gene and the internal URA3 gene when different concentrations ofPPR1 protein were expressed. The level of ura3-52 RNA was used as astandard for PPR1^(c) concentration in vivo in comparing the two URA3loci; ura3-52 has the same upstream sequences as URA3 and is responsiveover a wide range of PPR1^(c) concentrations. The level of GALPPR1-1expression was varied by growing cells with different concentrations ofgalactose in the medium; levels of ura3-52 RNA confirmed that higherconcentrations of galactose did in fact result in higher intracellularPPR1^(c) protein concentrations.

The results show that URA3 at the telomeric locus was less responsive tolow levels of the transactivator than URA3 at an internal locus. Inaddition, while both loci can achieve the same maximum level ofexpression, a higher PPR1^(c) concentration was required for thetelomeric URA3 compared to the internal URA3. These results suggest thatthere is a competition for binding at the telomeric URA3 promoterbetween PPR1^(c) and silent chromatin.

5. PPR1^(c) Activation of a Telomeric URA3 Gene is Cell Cycle Regulated

The studies described above were performed on actively dividing cells.Hence, the cells were transiting through the cell cycle during theanalysis. Keeping this in mind, two simple models can be set forth toexplain the competition between PPR1 and telomeric chromatin forexpression of the URA3 gene. In the first model, the competition onlyoccurs within specific periods of the cell cycle. During part of thecell cycle the telomeric URA3 gene is resistant to activation by PPR1 ifthe silent chromatin state has been established. Only when the silentchromatin is weakened or disassembled, which might occur during DNAreplication of the telomeric region, does PPR1 have the opportunity toactivate the gene. In the second model, PPR1 competes with equal fervorthroughout the cell cycle.

To test and distinguish between these models, cells were grown in richmedium containing 3% raffinose and no galactose. Thus PPR1^(c) was notpresent and the telomeric URA3 gene was maintained in a silent state.The cells were then synchronously arrested by treatment with eitherα-factor pheromone, to arrest them late in G₁ (Pringle and Hartwell,1981), or nocodazole, an inhibitor of microtubule assembly (Pillus andSolomon, 1986).

In many eukaryotes, nocodazole produces a synchronous arrest atmetaphase. Nocodazole also produces a very synchronous arrest in yeast,however it is unclear whether the arrest occurs late in G₂ or atmetaphase. By the criterion of spindle pole body separation the cellsappear to be in G₂ (Jacobs et al., 1988); however recent studies suggestthat the chromosomes may be condensed as expected for a metaphase arrest(Guacci et al., 1994). In light of this uncertainty, the arrest isreferred to as G₂/metaphase. Once arrested, galactose was added toinduce expression of PPR1-1, and half of the culture was released fromthe arrest, while arrest was maintained in the other half.

Expression of the telomeric URA3 gene and the internal URA1 and ura3-52genes was compared. The transcript levels of CLN2 and SWI5 were alsoanalyzed to monitor the progress of cells through the cell cycle. CLN2is transiently expressed in late G1 near the time of START (Wittenberget al., 1990), and SWI5 is transiently expressed beginning sometime inS, through G₂, and on into M (Nasmyth et al., 1987).

The telomeric URA3 was not activated by PPR1^(c) during α-factor arrest.The analysis clearly shows that while cells were arrested with α-factor,the telomeric URA3 gene remained repressed. The increase in URA1 andura3-52 mRNA levels indicate that PPR1^(c) was active in these cells.Following release from the α-factor arrest, PPR1^(c) was able toactivate the telomeric URA3 gene. The analysis of the SWI5 transcriptand microscopic analysis of cell morphology were consistent with thecell-cycle arrest imposed by α-factor, and release thereafter. The lowlevel of telomeric URA3 transcript seen late during the continuedα-factor arrest correlated with the small fraction of cells (˜5%) thatescaped from the arrest.

In striking contrast to the repressed state of telomeric URA3 duringα-factor arrest, the telomeric URA3 gene in G₂/metaphase, nocodazolearrested, cells was effectively activated by PPR1^(c). In the absence offunctional PPR1^(c), “GALppr1-1”), no activation of the telomeric URA3or the internal URA1 and ura3-52 genes occurred. In fact, not even basalexpression of the telomeric URA3 was seen in the absence of PPR1^(c).Analyses of CLN2 and SWI5 expression, as well as microscopic analyses ofcell morphology, confirmed the successful arrest with nocodazole and therelease that followed.

To determine whether the effects of the α-factor and nocodazoletreatments were due to the specific cell cycle arrests and not to otherphysiological effects of the treatments, the inventors tested the effectof α-factor on telomeric gene expression in cells arrested in G₂ withnocodazole, and conversely, the effect of nocodazole on telomeric geneexpression in cells arrested in G₁ with α-factor.

The α-factor treatment did not prevent the expression of the telomericURA3 gene in cells previously arrested with nocodazole, and nocodazoletreatment did not result in expression of the telomeric gene in cellspreviously arrested with α-factor. Thus, it appears that the effects ontelomeric gene transcription by α-factor and nocodazole were due to thespecific cell cycle arrests. These results suggest that the ability of atransactivator (PPR1^(c)) to function in a telomeric domain is cellcycle regulated. The inventors propose that a transactivator isinaccessible to the telomeric domain in G₁ phase and becomes accessibleby the time the cells are in G₂/metaphase.

To more accurately determine the period of the cell cycle in whichPPR1^(c) activation of a telomeric URA3 could occur, cells were arrestedin S phase with hydroxyurea, an inhibitor of DNA replication (Slater,1973). Yeast cells with a telomeric URA3 and the integrated GALPPR1-1fusion were pregrown in medium lacking galactose, to maintain repressionof the telomeric URA3 gene, and arrested with α-factor. Galactose wasadded to the α-factor arrested cells to induce expression of PPR1-1, andthe cells were released from the α-factor arrest; half of the culturewas released into medium containing hydroxyurea.

Cells treated with this α-factor/hydroxyurea protocol arrest very earlyin S phase, significantly before telomeric regions replicate (Hartwell,1976; McCarroll and Fangman, 1988). Hydroxyurea prevented the activationof the telomeric URA3, but did not affect transcriptional activation ofthe internal URAL and ura3-52 genes. Telomeric URA3 and SWI5 expressionfollowing release from the hydroxyurea arrest, indicated that the arrestwas reversible. Additionally, hydroxyurea did not prevent activation ofthe telomeric URA3 gene in cells which were previously arrested inG₂/metaphase with nocodazole, indicating that the presence ofhydroxyurea itself does not prevent telomeric URA3 expression. Theseresults indicate that early in S phase the transactivator can not gainaccess to the telomeric URA3, and taken together with the results above,suggest that progression through S phase is required for theestablishment of the transcriptionally active state in the telomericdomain.

Temperature sensitive alleles of CDC (Cell Division Control) genesrepresent another method commonly used to arrest yeast cells at aspecific point in the cell cycle (Pringle and Hartwell, 1981). Cells aretypically shifted from a permissive growth temperature (˜23°) to anon-permissive temperature (37°) to cause arrest. The inventors began touse temperature sensitive alleles of CDC genes to define the cell cycleperiod in which PPR1 activation occurred. However, it was discoveredthat PPR1^(c)-induced expression of a telomeric URA3 was severelycompromised at 37° in wild type (CDC⁺) cells (Aparicio, 1993). Thisfinding precluded the use of temperature sensitive alleles in dissectingthe period of activation in the cell cycle. The effect appeared to betelomere specific, since the ura3-52 locus was activated. It is notclear if the effect of temperature on telomeric URA3 activation wasspecific to PPR1^(c) (e.g. a reduction in the effective concentration ofPPR1^(c)), or reflects a general strengthening of telomeric repression.

6. Telomeric Silencing is Irreversible When Cells Are in StationaryPhase (G₀)

An additional means to synchronously arrest a population of yeast cellsis to maintain a culture in stationary phase (Werner-Washburne et al.,1993, for a review). Stationary phase cells of S. cerevisiae arrest in astate referred to as G₀;the cells are unbudded and their genomes areunreplicated. Cells enter G₀ by exiting from G₁ phase, and generaltranscriptional repression occurs upon entry to stationary phase(Choder, 1991).

Strains with URA3 at a telomeric or a nontelomeric locus and anintegrated GALPPR1-1 were grown to stationary phase in rich mediumcontaining 3% raffinose, so that PPR1^(c) was absent and hence thetelomeric URA3 gene was silenced. Cells were determined to be instationary phase when the optical density of the culture had notincreased during the previous 24 hour period, and greater than 98% ofcells were unbudded. Expression of GALPPR1-1 was induced in thestationary cells by adding 0.3% galactose to the cultures. Incubationwas continued as aliquots were collected for RNA analysis.

While the internal URA3 gene, as well as the URA1 and ura3-52 genes weretranscriptionally activated by PPR1^(c) in the stationary cells, thetelomeric URA3 gene was not activated. Only after 48 hours of inductionwas a telomeric URA3 transcript observed, just slightly above limits ofdetection. Thus, silencing of a telomeric gene in stationary phase cellsis essentially irreversible. As expected, basal levels of transcriptiondecreased in the stationary cells. Moreover, the SWI5 transcript was notdetected in Go cells, confirming that cells were not progressing throughthe mitotic cell cycle. In this study, galactose was added to culturesabout 48 hours after mid-log phase; equivalent results were obtainedwhen the study was performed with seven day old cultures.

C. Discussion

In this Example, the inventors examined the ability of transactivatorproteins to overcome silencing of a telomere-adjacent gene in S.cerevisiae. It was found that the transactivator protein, PPR1, isabsolutely required for expression of a URA3 gene located immediatelyadjacent to the left telomere of chromosome VII. In contrast, when URA3is at a non-telomeric location, PPR1 merely provides a modest increasein expression (Roy et al., 1990). Two conclusions may be drawn fromthese results: telomeres inhibit basal transcription, andtransactivators have a mechanism to circumvent this inhibition.

It is likely that the basal transcription apparatus of URA3 is preventedfrom accessing the gene's promoter due to steric occlusion by silenttelomeric chromatin. This is supported by the observation that other DNAbinding proteins, such as E. coli dam methylase, are excluded fromtelomere-proximal DNA regions in vivo (Gottschling, 1992). Note thatbasal expression of URA3, as with most housekeeping genes in yeast,requires not only a TATA element but additional sequences upstream thatbind PPR1-independent factors (Roy et al., 1990).

These results show that, first, PPR1 cannot activate transcription ofthe telomeric URA3 gene in G₁, early S, or G₀ cells. Only in aG₂/metaphase arrest is activation observed. Second, the cellularconcentration of PPR1 dramatically affects the frequency with whichtelomeric URA3 expression is established. Third, the complete activationdomain of a transactivator is essential for its efficacy. While atelomeric gene with a GAL4 UAS can be activated in the presence of wildtype GAL4, the gene remains silenced when the wild type GAL4 is replacedby derivatives which remove the GAL4 transcriptional activation domain.

The inventors propose a replication-dependent model to explain how atelomeric gene can overcome silencing to become transcriptionallyactive. In G₁ of the cell cycle, a silenced telomeric gene is packagedin a repressive chromatin structure which is relatively “static” andprevents interactions of the DNA with other DNA binding proteins such asbasal transcription factors and transactivators. However, the telomericchromatin loses its static structure, as a result of the DNA replicationprocess or some other coordinate cellular event. Alternatively, one ofthe two newly replicated sister chromatids retains the silent chromatinwhile the other is essentially ‘naked’ DNA and awaits assembly intochromatin.

Regardless of which pathway occurs, upon completion of replication, twodistinct assembly processes compete to establish the transcriptionalstate of a telomeric gene. Assembly of silent chromatin initiates at thetelomere and propagates inward along the DNA. This process requires notonly the histones but a number of additional factors, such as RAP1,SIR2, SIR3, and SIR4 (Example II; Kyrion et al., 1993). The competingprocess involves the binding of the transactivator protein to thetelomeric gene and assembly of an active transcription complex. Thecompetition ends when one of the two processes is fully established atthe promoter region of the telomeric gene. In the absence of competitionfrom the transactivator, the silent chromatin eventually assembles intoits static structure. The moment that this silent structure forms,defines the end of the cell cycle period in which the transactivator hasan opportunity to compete.

Having a limited period in the cell cycle during which a transcriptionalstate is established has several ramifications. Environmental or geneticchanges that alter the length of the silent chromatin assembly processcould dramatically affect the frequency of establishing a state. Suchchanges may be direct. For instance, the SIR3 gene product appears to bea component of silent chromatin that is rate-limiting in its assembly(Johnson et al., 1990; Example III). Thus increasing SIR3 concentrationincreases the frequency of establishing repression (Example III).Alternatively, changes that extend periods of the cell cycle in whichsilent chromatin assembly occurs, such as G₂, provide a transactivatorgreater opportunity to establish an active state. Conversely, a shorterG₂ would favor establishment of a silent state. In essence, such changescan dictate the amount of phenotypic variegation within a population ofcells.

The assembly of silent telomeric chromatin may consist of severaldistinct, sequential steps rather than an ‘all-or-none’, concertedprocess. In nocodazole-arrested cells, telomeric URA3 expression wasrapid when PPR1 was present (GALPPR1). However, basal, orPPR1-independent (GALppr1), expression of the telomeric URA3 was notdetected, even after a lengthy arrest (˜5 hr); while basal expression atinternal loci was normal. These results suggest that at thenocodazole-arrest point silent chromatin is assembled up to a stage thatprecludes basal expression, yet does not prevent PPR1-inducedexpression.

This postulated intermediate of silent chromatin assembly may not belocked into a fully static structure, yet it is still more recalcitrantto gene expression than other areas of the genome. The static chromatinstructure likely requires several contributions: binding of the corehistones by accessory proteins such as SIR3 (Example III) modificationsof telomeric histones such as hypoacetylation (Braunstein et al., 1993),and localization of the structure to the nuclear periphery (Palladino etal., 1993). Any of these contributions may be absent at an intermediatestage.

These results extend observations made at the yeast silent mating typeloci, HML and HMR (Miller and Nasmyth, 1984). Telomeres and the HM locishare a number of silencing factors (e.g. SIR2, SIR3, and SIR4) andNasmyth determined, using temperature sensitive alleles of SIR3 andSIR4, that establishment of silencing at the HM loci requires passagethrough S phase, and thus presumably DNA replication. Their conclusionis consistent with the model the inventors propose, that the competitionfor assembly occurs after replication. Furthermore, the inventors showthat, at least in the case of the VII-L telomeric locus, assembly ofsilencing is not completed until sometime after G₂/metaphase(nocodazole-arrest).

Miller and Nasmyth also found that inactivating the SIR3 or SIR4 geneproduct at any time in the cell cycle resulted in gene expression at theHM loci. Here, the inventors show that passage through S phase isrequired for activation of a telomeric gene. Thus, dismantling of therepressive chromatin, either by artificially compromising it with adefective SIR3 or SIR4 allele, or in every cell cycle during passagethrough S phase, allows a renewal of the competition betweenestablishment of active and silent states.

As the result of a telomeric location, URA3 can be much more highlyregulated than at its normal locus. When URA3 is at a non-telomericlocation, the presence of PPR1^(c) produces a three to seven-foldinduction over basal expression (Liljelund et al., 1984). However, withURA3 near a telomere, an equivalent amount of PPR1 induces expressionabout 100-fold. The inventors suggest that the genomes may have evolvedto take advantage of this type of telomeric regulation. For example,Trypanosomes depend upon the highly regulated expression of thetelomeric VSG (Variable Surface Glycoprotein) genes (Borst, 1991, Cross,1990).

When cells were in G₀, essentially no amount of transactivator proteinwas sufficient to overcome telomeric silencing, while at an internalnon-silenced position the transactivator readily induced expression.Interestingly, general transcriptional repression, apparently mediatedby chromatin changes, occurs upon entry to stationary phase (Choder,1991). In fact, stationary phase chromosomes display differentsedimentation properties than G₁ phase chromosomes, suggesting thatchromosomes assume a distinct compact structure in G₀ cells (Pifion,1978). It is possible that the same machinery and mechanism of telomericsilencing in G₁ extends to other regions of the genome in G₀, thusfacilitating the more global compaction and transcriptional repression.

Whatever the nature of the silent telomeric chromatin, it contrasts withthe chromatin structure of the PHO5 gene in yeast. While this locus istranscriptionally repressed by nucleosomes upstream of the transcriptioninitiation site, it can be induced rapidly at anytime in the cell cycleor in G₀ arrested cells (Schmid et al., 1992). The induction involvesthe displacement of a nucleosome by the gene's transcriptional activatorprotein. In contrast, overcoming telomeric silencing requires that thenucleosomes be modified or removed by passage through S phase before thetransactivator protein can have its effect. This emphasizes thattelomeric chromatin is inherently different than chromatin at PHO5 ormost other regions of the yeast genome.

EXAMPLE V Identification of Genes that Suppress Telomeric Silencing

Genes located near S. cerevisiae telomeres are subject totranscriptional silencing by a repressive chromatin structure thatinitiates at the telomeres (Gottschling et al., 1990; Gottschling, 1992;Renauld et al., 1993; Examples I through IV). The inventors hypothesizedthat the telomeric structure responsible for silencing is likely to be amultimeric complex that would be sensitive to the stoichiometricimbalance of its components. Therefore, in order to identify genesinvolved in telomere structure or function, the inventors carried out ascreen for gene products that, when expressed at high levels, wouldsuppress telomeric silencing.

A yeast strain was constructed with genetic markers located at twotelomeric loci. The ADE2 gene, which is required for adeninebiosynthesis, was placed adjacent to the telomere at the right arm ofchromosome V (V-R), and URA3, a gene required for uracil biosynthesis,was located adjacent to the telomere at the left arm of chromosome VII(VII-L).

More specifically, the strain used for transformation with the librarywas UCC3505 (MATa ura3-52 lys2-801 ade2-101 trp1-Δ63, his3-Δ200 leu2-Δ1ppr1::HIS3 adh4::URA3-TEL DIA5-1). DIA5-1 refers to the directedintegration of ADE2 adjacent to telomere V-R. UCC3505 was constructed bysuccessively transforming YPH499 (Sikorski & Hieter, 1989) with pVII-LURA3-TEL (Gottschling et al., 1990), pΔPPR1-HIS3 (Renauld et al., 1993),and pHR10-6. Plasmid pHR10-6, obtained from H. Renauld, was constructedby inserting a 2.8 kb Hind III fragment from plasmid pV-R URA3-TEL(Gottschling et al., 1990), containing sequences from the subtelomericregion of chromosome arm V-R, into the Hind III site of pYTCA-2(Gottschling et al., 1990), such that the Eco RI site of the insert wasfurthest from the Bam HI site of the vector, thus creating pHR9-9. Intothe Bam HI site of pHR9-9 was inserted the 3.4 kb Eco RI-Bam HI fragmentcontaining the ADE2 gene from pL909 (Gottschling et al., 1990), thuscreating pHR10-6. The ADE2 gene is oriented with its promoter proximalto the V-R sequences. pHR10-6 was cleaved with Eco RI for use infragment-mediated transformation of yeast.

Normally, colonies expressing ADE2 are white, while those not expressingit (ade2) are red (Roman, 1956). Due to the semi-stable nature oftelomeric silencing of most genes, switching between silenced andtranscriptionally active states may occur every few generations, givingrise to different phenotypic populations. In the case of strains withADE2 near a telomere, these different populations are seen as red andwhite sectors within a single colony (Gottschling et al., 1990). A URA3gene located at telomere VII-L also normally switches betweentranscriptional states (Gottschling et al., 1990). However, thetelomeric URA3 was caused to be completely silenced by deleting itstrans-activator, PPR1 (Aparicio & Gottschling, 1994). The cells weretherefore unable to grow in the absence of uracil.

To identify genes or gene fragments whose overexpression could disruptsilencing, the strain was transformed with a high-expression S.cerevisiae cDNA library. The pTRP plasmid expression library used inthis study was created with cre-lox site-directed recombination from theXTRP library (obtained from S. J. Elledge, Baylor College of Medicine,Houston). The pTRP vector contains a 2μ origin of replication and theTRP1 selectable marker. The cDNA inserts were cloned into a Xho I siteof the pTRP vector, placing them under the control of the GAL1 promoter.The creation of similar libraries is described in Elledge et al. (1991).

By the nature of its synthesis, a cDNA library typically contains bothfull length and truncated versions of RNA transcripts. Thus high levelexpression from a cDNA library has two means of causing a stoichiometricimbalance: by expression of a normal gene product or a defective one(Herskowitz, 1987). In the library used in this study, the expression ofcDNA inserts was controlled by the GAL1 promoter, which is stronglyinduced by the presence of galactose in the medium (Johnston & Davis,1984). Of the 330,000 yeast transformants obtained, 48 displayed agalactose-dependent decrease in telomeric silencing. That is, when grownon media containing galactose, the cells were able to grow in theabsence of uracil (Ura⁺) and gave rise to predominantly white colonies(Ade⁺). On the basis of restriction mapping, DNA blotting (Southern)analysis, and DNA sequencing, it was determined that these 48 clonesrepresented ten independent genes.

EXAMPLE VI Isolation of TLC1, a Telomere-Specific Suppressor ofSilencing

The genes known to be required for telomeric silencing are also involvedin transcriptional silencing at two internal chromosomal sites, the HMLand HMR loci, which harbor the unexpressed copies of the mating typegenes in S. cerevisiae (Aparicio et al., 1991). To determine whether thenewly isolated suppressors of telomeric silencing also affect silencingat HML, the expression plasmids were introduced into a strain in whichthe URA3 gene was inserted into the HML locus. The strain used forassaying silencing at the HML locus was UCC3515 (MATα lys2-801 ade2-101trp1-Δ63 his3-Δ200 leu2-Δ1 ura3-52 hml::URA3). The hml::URA3 constructis the same as that described for strain GJY5 (Mahoney & Broach, 1989).Overexpression of one of the novel genes identified, TLC1, had no effecton silencing at HML, but strongly suppressed telomeric silencing of URA3and ADE2 (FIG. 1A, FIG. 1B and FIG. 1C). The SIR4 gene, whoseoverexpression disrupts silencing both at telomeres and at HML (Marshallet al., 1987), was also isolated in the present screen and derepressedboth of these loci in this assay (FIG. 1A, FIG. 1B and FIG. 1C).

Further evidence for the specific association of TLC1 with telomerestructure came from examination of telomere length in strainsoverexpressing a TLC1 cDNA clone. In the absence of the TLC1overexpression plasmid, the telomeric sequences at VII-L averaged 330base pairs (bp) in length. Upon overexpression of TLC1, the averagetelomere length at VII-L decreased between 90 and 220 bp (FIG. 2). Thealteration of telomere length upon overexpression of TLC1, together withthe loss of telomeric silencing, suggested that this gene isspecifically involved in telomere structure.

Of the 48 cDNA clones isolated in the present screen as suppressors oftelomeric silencing, nine represented TLC1. The inventors sequenced oneof the TLC1 cDNA clones in its entirety (pTRP61, 1248 bp), as well asthe ends of the other eight TLC1 clones. These sequence data overlappedto yield a contiguous sequence of 1301 bp, although no single cloneincluded the entire sequence. The combined sequence of the TLC1 cDNAclones has been submitted to GenBank and assigned the accession numberU14595.

The span of each of the cDNA clones with respect to the entire 1301 bpfragment is as follows: pTRP6 (1-1248), pTRP61 (54-1301), pTRP14 andpTRP47 (54-1263), pTRP33 and pTRP39 (54-1269), pTRP55 (54-1264 or 1265),pTRP59 (39-1250), pTRP60 (270-1264 or 1265), and pTRP61 (54-1301). Fourof the TLC1 cDNA sequences (in clones pTRP55, pTRP60, pTRP33 and pTRP39)are followed by short stretches (5-20 nts) of adenines. It is not yetclear whether these adenines reflect authentic in vivo polyadenylationof the TLC1 transcripts, or are by-products of cDNA synthesis.

For reference, both the TLC1 gene and the RNA template include theCACCACACCCACACAC (SEQ ID NO:3) template sequence that ultimately allowsthe GTGTGTGGGTGTG sequence (SEQ ID NO:2) to be inserted into thetelomere. The TLC1 gene sequence CACCACACCCACACAC (SEQ ID NO:3) spansthe region 468-483 of SEQ ID NO:1. In the complementary strand, SEQ IDNO:4, this region is 819-834.

Physical mapping localized TLC1 to a single site on chromosome II,immediately adjacent to CSG2. TLC1 was mapped by hybridizing the labeledcDNA clone (1.25 kb Xho I insert from pTRP6) to a filter grid containingλ phage clones representing over 96 percent of the yeast genome. Thefilter set was obtained from the American Type Culture Collection (Olsonet al., 1986; Link & Olson, 1991; Beeler et al., 1994). Subsequent tothe present work, the sequence of chromosome II was entered into theEMBL database. The chromosome II-R sequences have the EMBL accessionnumber X76078. These data matched the present sequence obtained from thecDNAs.

RNA blot (Northern) analysis confirmed that a wild-type strain containeda relatively abundant RNA that hybridized to a TLC1 probe and wasapproximately 1.3 kilobases (kb) in length (FIG. 3A and FIG. 3B).

EXAMPLE VII TLC1 Encodes the Telomerase RNA

The TLC1 sequence has two notable features. The gene is unlikely toencode a protein since it does not contain a large open reading frame(ORF). The longest ORF that begins with an ATG codon is only 43 aminoacids in length. This finding suggested that the functional TLC1 geneproduct might be the RNA itself. Moreover, TLC1 contains the sequenceCACCACACCCACACAC (SEQ ID NO:3), which includes the motif predicted totemplate S. cerevisiae telomeres (Kramer & Haber, 1993). These resultssuggested to the inventors that TLC1 encodes the putative yeasttelomerase RNA.

To confirm that the TLC1 gene product is indeed the telomerase RNA, theTLC1 gene was disrupted. The inventors predicted that this would causeincomplete replication of telomeres and result in progressive telomereshortening with each cell division. A TLC1 gene disruption was createdin which a large part of TLC1, including the predictedtelomere-templating region, was removed and replaced with a marker gene.

For the gene disruption, the TLC1 cDNA clone in plasmid pTRP61 wasexcised away from pTRP vector sequences as a 1.25 kb Xho I fragment, andinserted into the Xho I site of pBluescript II KS(−) (Stratagene; LaJolla, Calif.), creating pBlue61. The disruption of TLC1 was created byreplacing the 693 bp Nco I-Nsi I fragment of pBlue61 with a blunt-endedBam HI 1.6 kb LEU2 clone from plasmid YDp-L (Berben et al., 1991),creating pBlue61::LEU2. This construct was digested with Xho I andtransformed into the diploid strain UCC3507, selecting for Leu⁺transformants, to produce UCC3508 (UCC3507 TLC1/tlc1::LEU2). Southernblot analysis confirmed that UCC3508 was heterozygous for the disruptionat the TLC1 locus.

Nineteen out of nineteen tetrads sporulated from UCC3508 yielded 2:2segregation of the tlc1::LEU2 allele. The genotype of UCC3507 is:MATa/MATa ura3-52/ura3-52 lys2-801/lys2-801 ade2-101/ade2-101his3-Δ200/his3-Δ200 trp1-Δ1/TRP1 leu2-Δ1/leu2-Δ1adh4::URA3-TEL/adh4::URA3-TEL DIA5-1/DIA5-1 ppr1::HIS3/ppr1::LYS2. Thehaploid strains crossed to create UCC3507 were derived from YPH250 andYPH102 (Sikorski & Hieter, 1989). The introduction of changes into thegenotypes of these haploids all utilized plasmids described above,except allele ppr1::LYS2, which was introduced using plasmidpΔPPR1::LYS2 (Renauld et al., 1993).

The disrupted gene was introduced into a wild-type diploid strain tocreate a TLC1/tlc1 heterozygote, which was then sporulated, giving riseto two mutant and two wild-type haploid strains. Northern analysisconfirmed that in the TLC1-disrupted spore products, there was nodetectable TLC1 RNA (FIG. 3A and FIG. 3B). The spore colonies wereinoculated into rich medium and grown for several days by diluting thecultures into fresh medium every 24 hours. In all cases examined (eighttetrads), TLC1 strains maintained a normal telomere length after 6 daysof growth. In contrast, the tlc1 strains displayed shortened telomeres.In the cases where DNA samples were collected daily (three tetrads), thetlc1 telomeres were found to shorten progressively, at an approximaterate of 3 bp per generation (FIG. 4A).

In conjunction with the shortening telomere phenotype, older tlc1cultures displayed a gradual increase in generation time. Through thefirst 40 generations after sporulation of a TLC1/tlc1 strain, all fourspore products were able to regrow approximately one thousand-fold inrich medium within 24 hours, indicating a generation time of less than2.4 hours (FIG. 4B). This growth rate was maintained in TLC1 strains forup to 80 generations.

In contrast, the tlc1 strains, by 65 generations after germination, thegrowth rate had slowed to about 3.3 hours/generation. After 75generations, the doubling time of the tlc1 cultures was 5.7 hours. Thisdecrease in growth rate was accompanied by a 50% drop in viability inthe tlc1 strains after 75 generations. This general pattern was clear inall 14 tetrads examined, although there was some variation in the periodat which the decrease in growth rate occurred. However, as was reportedfor estl strains (Lundblad & Blackburn, 1993), the dying tlc1 cultureswere overwhelmed within approximately 100 generations by faster-growingcells, which presumably contained suppressor mutations.

To demonstrate that the TLC1 gene product was the S. cerevisiaetelomerase template RNA, it was necessary to confirm that TLC1 sequencesencoded telomeric tract repeats. Earlier studies with Tetrahymenathermophila showed that when a mutated telomerase RNA is introduced intoa cell, the altered sequence may then be templated into the cell'stelomeres (Yu et al., 1990). A candidate motif for the telomere templatewithin TLC1 was the sequence CACCACACCCACACAC (SEQ ID NO:3) (FIG. 5A).The inventors constructed a TLC1 allele, designated TLC1-1(Hae III), inwhich two base pairs of this motif were changed to create a recognitionsite for the restriction enzyme Hae III (FIG. 5B).

The mutant TLC1-1(Hae III) allele was used to replace one of the normalTLC1 genes in a diploid strain as follows: Plasmid pVZ61b wasconstructed by inserting the 1.25 kb Xho I fragment containing the TLC1cDNA clone from pTRP61 into the Sal I site of plasmid pVZ1 (Henikoff &Eghtedarzadeh, 1987). The TLC1-1(Hae III) mutant allele was generatedusing two oligonucleotides, Hpa I primer(5′-TCCAGAGTTAACGATAAGATAGAC-3′) and Hae III primer (5′-TAATTACCATGGGAAGCCTA CCATCACCAGGCCCACACAC AAATG-3′; SEQ ID NO:5 [Greider andBlackburn, 1985, 1987, 1989; Zahler and Prescott, 1988; Morin, 1989;Prowse et al., 1993; Shippen-Lentz and Blackburn, 1989; Mantell andGreider, 1994; de Lange, 1994; Greider, 1994; Harley et al., 1992]) toPCR-amplify a 232-bp fragment from plasmid pVZ61b.

The PCR product was then cleaved with Nco I and Hpa I, to create a 213bp fragment that was used to replace the 213-bp Nco I-Hpa I fragment ofpBlue6l, to create pBlue61-Hae III. The 213 bp fragment was sequencedfrom the pBlue61 plasmid to verify that the PCR amplification did notintroduce additional mutations into the sequence.

The TLC1-1(Hae III) allele, contained in a 1.25 kb Xho I fragment, wasthen cleaved from pBlue6l-Hae III and inserted into the Xho I site ofpRS306 (Sikorski & Hieter, 1989), to create the integrating plasmidpRS306-TLCl-1(Hae III). This latter construct was digested with Afl IIand used to transform YPH501 (Sikorski & Hieter, 1989), with selectionfor Ura⁺ transformants, thus creating the heterozygous strain UCC3520.UCC3522 (YPH501 TLC1-1(Hae III)/TLC1) was isolated as a 5-fluoro-oroticacid-resistant derivative of UCC3520 in which the pRS306-TLC1 plasmidhad recombined out of the TLC1 locus, which left the TLC1-1(Hae III)allele in the chromosome (Scherer & Davis, 1979), as confirmed bySouthern blot analysis.

In addition to functioning at the very ends of normal telomeres,telomerase is also believed to play an important role in the healing ofbroken chromosomes and the extension of unusually short telomeric tracts(Kramer & Haber, 1993). In this latter capacity, the activity of amutant telomerase would be most easily detected. Therefore,fragment-mediated transformation was used to remove the sequence distalto the ADH4 locus on the left arm of chromosome VII, and replace it witha URA3 gene and a short tract of telomeric sequence to act as a seed forin vivo telomere elongation (FIG. 6A).

This transformation was done in both homozygous wild-type (TLC1/TLC1)and heterozygous TLC1-1(Hae III)/TLC1 strains. The ADH4-URA3-TG₁₋₃fragment used to replace the left arm of chromosome VII was generated byNot I-Sal I digestion of plasmid AD3ARUGT-IV. This plasmid wasconstructed by the following set of steps: the 1.1 kb Hind III-Sma I DNAfragment containing URA3 (Rose et al., 1984) was inserted into the HincII site of pYTCA-2 by blunt-end ligation, with the promoter of URA3proximal to the TG₁₋₃ sequences of the vector, creating plasmid p3ARUCA.The 1.2 kb Hind III fragment of pYA4-2, containing ADH4 (Lundblad &Szostak, 1989; Williamson & Paquin, 1987), was then inserted into theHind III site of p3ARUCA, with the Sal I site of the insert distal tothe URA3 gene in the vector, creating plasmid pAD3ARUCA. Finally, theSal I-EcoR I fragment containing the composite insert (ADH4-URA3-TG₁₋₃)from pAD3ARUCA was cloned into pVZ1, creating AD3ARUGT-IV.

The yeast strains that were transformed with the ADH4-URA3-TG₁₋₃fragment were YPH501 (TLC1/TLC1) and UCC3522 (TLC1-1(HaeIII)/TLC1).These studies were repeated with the transforming ADH4-URA3-TG₁₋₃ DNAliberated from the pAD3ARUGT-IV plasmid as a Sal I-EcoR I fragment, andresults similar to those reported in FIG. 6B were obtained.

Southern analysis was performed on genomic DNA from the transformedstrains to determine the structure of the new telomeres at VII-L (FIG.6B). Digestion with Apa I, whose most distal site in the new VII-L armoccurs within the URA3 gene, demonstrated that in both the wild-type(TLCL/TLC1) and heterozygous TLC1-1(Hae III)/TLC1 transformants, the newchromosomal end was extended in vivo to several hundred base pairs. Thenew telomeres in the TLC1-1(Hae III)/TLC1 strain were slightly shorterand more heterogeneous in length than those added in the TLC1/TLC1strain.

In all twelve TLC1/TLC1 independent transformants tested, digestion withHae III, which cuts at the same site in URA3 as Apa I, indicated that noHae III sites were introduced during telomere elongation in vivo. Incontrast, in all eight TLC1-1(Hae III)/TLC1 independent strainsexamined, Hae III sites were incorporated into the newly formedtelomere. It can thus be concluded that the mutated sequence in theTLC1-1(Hae III) gene served as a template for the addition of telomericrepeats, which indicates that the TLC1 gene indeed encodes the S.cerevisiae telomerase RNA.

EXAMPLE VIII TLC1 Compared to Other Telomerase RNAs

In these studies the inventors demonstrated the existence of an S.cerevisiae telomerase and identified the gene that encodes its RNAcomponent (Examples V through VII). These above findings support theproposal that the telomerase mechanism of replicating the ends ofchromosomes is widespread among eukaryotes. However, the TLC1 RNA ismuch larger (1.3 kb) than the known ciliate telomerase RNAs, which are160 to 200 nucleotides (nt) in length (Blackburn, 1993). Thisdiscrepancy in gene size is reminiscent of the 1175 nt S. cerevisiae U2snRNA, which is almost 1 kb larger than the mammalian U2 snRNA (Ares,1986). The conserved secondary structure that is shared among theciliate telomerase RNAs is not apparent in the sequences surrounding theTLC1 template region (Romero & Blackburn, 1991; ten Dam et al., 1991),though the large size of the transcript may allow homologous structuresto form that are not obvious at this time. TLC1 also lacks a shortprimary sequence adjacent to the template region that is conserved amongthe ciliate telomerase RNAs (Lingner et al., 1994).

While telomeric DNA in most organisms is comprised of sequences repeatedin a regular fashion, e.g. mammalian (T₂AG₃), Tetrahymena (T₂G₄), thetelomeric sequence of S. cerevisiae is irregular [(TG)₁₋₃TG₂₋₃] (Zakian,1989). However, this irregularity can be fully explained by thetelomere-templating sequence in TLC1. Telomerase RNAs are thought tosynthesize the G-rich strand of telomeres by multiple rounds ofhybridization to a short sequence at the end of a telomeric tract,elongation of the DNA by a limited reverse transcription of the RNA, anddisengagement (Blackburn, 1993). In vitro, the Tetrahymena telomeraseRNA appears to use as few as three nucleotides for the hybridizationstep (Autexier & Greider, 1994).

The telomere template region of TLC1 (CACCACACCCACACAC; SEQ ID NO:3)suggests that the telomerase RNA may be able to align with a telomereterminus at a number of different points within the RNA, especially ifCAC is all that is required for hybridization. It is also possible thatthe telomerase could abort a round of reverse-transcription at severaldifferent positions along the RNA. If a terminal DNA sequence such asGTG is left, then alignment with the CAC RNA motif in the next round ofelongation can readily occur. Either alone or in combination, thesedifferent alignment and termination possibilities can account for theheterogeneity observed in the S. cerevisiae telomeric tracts.

EXAMPLE IX Telomeric Silencing and Telomerase

Overexpression of the TLC1 cDNA clones identified in the present studies(Examples V through VIII) both disrupts telomeric silencing and causes ashortening of telomeres. One model to explain these results is thatoverexpression of the cDNAs causes limiting telomerase components to betitrated into incomplete and nonfunctional complexes, thereby reducingthe total telomerase activity in the cell and resulting in shortertelomeres. The length of the telomere may relate to its ability to bindsilencing proteins; shorter telomeres simply have fewer binding sites,and thus may silence telomeric genes less efficiently (Kyrion et al.,1993). Alternatively, the telomerase RNA itself, or one of the factorsit binds, may be an integral component of the complex that is requiredfor silencing at telomeres. Overexpression of TLC1 may perturb thestoichiometry of this complex, and thus interfere with its assembly. Itis noteworthy that of the nine TLC1 cDNAs isolated in the presentscreen, none appear to be full length. Thus it is formally possible thatonly an incomplete (non-functional) TLC1 RNA can produce the effectsdetected.

The telomere shortening and growth defects observed when the telomeraseRNA was disrupted are very similar to those described for estl strains,supporting the prediction that EST1 is a constituent of telomerase(Lundblad & Szostak, 1989). Moreover, the genetic link discovered herebetween telomeric silencing and telomerase suggests future approachesfor identifying other telomerase components, which so far have beenelusive.

EXAMPLE X Other Genes Identified by Telomeric Silencing

Using the telomeric silencing protocol described herein, the inventorsisolated 48 clones. On the basis of restriction mapping, DNA blotting(Southern) analysis, and DNA sequencing, it was determined that these 48clones represented ten independent genes. Of the these ten genes, fourhave been sequenced and identified previously. These genes are the SIR4(Marshall et. al., 1987); ASF1 (Le and Sternglanz, Genbank AccessionNumber 107593); RPL32 (Dabeva and Warner, 1987); and RRP3 (Cherel andThuriaux, Genbank Accession Number z29488).

The new genes are herein termed STR genes, Suppressors of TelomericRepression. Initially, seven STR genes were designated, although STR7was later found to correspond to part of the sequence for RRP3. STR2 hasbeen renamed TLC1 following its functional characterization, as shown inFIG. 7A and FIG. 7B.

The DNA and predicted amino acid sequences, where relevant, of the STRgenes are as defined in Table 2.

TABLE 2 Probes & Primers DNA Complementary Projected SEQ StrandPolypeptide SEQ ID Gene ID NO: SEQ ID NO: SEQ ID NO: NOS: STR1 15 29† 165837-7702 TLC1 (STR2)  1  4 *  33-1317 STR3 17 30† 18 7703-8780 STR4 19†20 1318-3735 STR5 21 31† 22 8781-9571 STR6 23† 24 3736-5836 STR7 25 32†26 RRP3 27\ 28 *Encodes RNA Template - SEQ ID NO:3 554 Denotes strandwith protein-encoding open reading frame

Table 2 shows the DNA and amino acid sequences of seven of the STRgenes. STR2, renamed TLC1, encodes the RNA template component, ratherthan a polypeptide species. Both SEQ ID NO;1 and SEQ ID NO:4 areprovided for TLC1. STR7 (SEQ ID NO:25, DNA; and SEQ ID NO:26, aminoacid) was found to be a partial sequence of RRP3, the full lengthsequences of which are also included herein (SEQ ID NO:27, DNA; and SEQID NO:28, amino acid).

Table 2 also provides information concerning the numbers of 17-merprobes and primers from SEQ ID NO:1 and from each of thepolypeptide-encoding DNA sequences of the present invention. Naturally,the number of 17-mers from each of the complementary strands could bereadily made. Given that 32 separate sequences are already disclosedherein, should the 17-mer probes and primers from the claimed sequencesbe specifically identified and numbered, they would start with SEQ IDNO:33.

The projected SEQ ID NO designations in Table 2 refer to the individualsequences that could be readily predicted from the given information.For example, the sequence AATAAAACTAGAGAGGA, residues 1 to 17 of SEQ IDNO:1, would be assigned SEQ ID NO:33; the sequence ATAAAACTAGAGAGGAA,residues 2 to 18 of SEQ ID NO:1, would be assigned SEQ ID NO:34. On thisbasis, SEQ ID NO:100 would be ATTTTTTTTTTTTTCAG, residues 68 to 84 ofSEQ ID NO:1; SEQ ID NO:1000 would be GATCAAGAACGTAATTT, residues 968 to984 of SEQ ID NO:1; SEQ ID NO:5000 would be AAAAGATGAAGACGCTT, residues1265 to 1281 of SEQ ID NO:23; and SEQ ID NO:9571 would beAGATATTCTAACTCTCT, residues 791 to 807 of SEQ ID NO:31.

The start and stop site locations for the major open reading frames(ORFs) of each of the STR sequences are presented in Table 3. The ORFsfor STR4 and STR6 are presented with respect to the DNA strandoriginally sequenced. It was noted that certain of the DNA sequences hadORFs oriented in the opposite direction to the original DNA strandsequence, so that the ORF starts at a high position in the DNA, and endsat a low position. Namely, the STR1 ORF was located between nucleotides1829-84; the STR3 ORF was located between nucleotides 1017-1; and theSTR5 ORF was located between nucleotides 753-109. Although thisphenomenon is well known, the complementary DNA strand of STR1, STR3,STR5 and STR7 are also included herein (SEQ ID NO:29, SEQ ID NO:30, SEQID NO:31 AND SEQ ID NO:32, respectively; Table 2 and Table 3), and theORFs listed in ascending numbers for instant recognition.

TABLE 3 Length of Original ORF Strand Complementary ORF (Amino SEQ IDStrand Starts ORF Ends Acid Gene NO: SEQ ID NO: at (bp #) at (bp#)Residues) STR1 15 29\ 54 1799 582 STR3 17 30\ 78 1094 339 STR4 19\  22368   789 STR5 21 31\ 55  699 215 STR6 23\  3 1955   651 STR7 25 32\279   956 226 †Denotes strand with open reading frame (ORF)

To determine how strongly each gene suppressed telomeric silencing,viability in the absence of uracil was quantified for the strains thatcontained the telomeric URA3 gene and each of the highly expressedgenes. All the genes suppressed silencing of the telomeric URA3,although a hierarchy of suppression was observed (FIG. 7A).

All previously identified genes known to be required for telomericsilencing are also known to be involved in transcriptional silencing attwo internal chromosomal sites, the HML and HMR loci, which harbor theunexpressed copies of the mating type genes in S. cerevisiae (Aparicioet al., 1991). To determine whether the newly isolated suppressors oftelomeric silencing also affect silencing at HML, the expressionplasmids were introduced into a strain in which the URA3 gene wasinserted into the HML locus (Mahoney & Broach, 1989).

Overexpression of the novel gene TLC1 (STR2) had no effect on silencingat HML, but strongly suppressed telomeric silencing of URA3 and ADE2(FIG. 7B). The SIR4 and ASF1 genes, whose overexpression was previouslyknown to disrupt silencing both at telomeres and at HML (Marshall etal., 1987), as well as STR1, STR4, and RRP3 genes, derepressed HML verywell (FIG. 7B). Overexpression of RPL32, STR3, STR5 and STR6 hadintermediate effects at HML (FIG. 7B).

EXAMPLE XI Detailed Analysis of the TLC1 Gene and RNA

To define the components of telomerase activity, the telomerase templateRNA from S. cerevisiae is used in conjunction with classical andmolecular genetic techniques to identify the previously elusivetelomerase proteins.

Telomere length in S. cerevisiae is normally under tight geneticcontrol; telomeres do not grow infinitely long, nor do they becomedrastically shortened. In addition, a 3′ tail is detected at the end ofyeast chromosomes late in S-phase. Taken together these observationssuggest that telomerase activity is regulated, most likely being limitedto late S-phase of the cell cycle. There are numerous mechanisms toexplain the proposed modulation of telomerase activity in a cell. At afirst level of evaluation the models can be divided into those in which(1) the RNA is regulated (the RNA is the limiting component), (2) adifferent component of telomerase activity (mostly likely a protein) isregulated, or (3) that telomerase activity is constitutive and access toits substrate (the 3′ end of the chromosome) is regulated. Validity ofthese models concerning telomerase regulation is determined as follows.

A. Fine structure Analysis of the TLC1 RNA

To determine the 5′ and 3′ ends of the telomerase RNA, standardtechniques, such as Si and ribonuclease protection (for both the 5′ and3′ ends), and primer extension (for the 5′ end) are used (Sambrook etal., 1989). By using this combination of methods, the physical ends ofthe RNA are identified.

Typically, non-translated RNAs do not have a polyA⁺ tail. However, ofthe nine TLC1 cDNA clones isolated in the earlier geneticselection/screen, four had adenine tracts of 5-20 nucleotides at their3′ ends. A recently published method is available for determining theprecise sequence of 3′ ends of messages, irrespective of whether theyhave a long, a short or no poly-A tail (Liu & Gorovsky, 1993). Themethod uses T4 RNA ligase to attach a DNA oligonucleotide to the 3′ endof RNA molecules, followed by cDNA synthesis, PCR amplification, cloningand sequencing. This method is capable of detecting a poly-A⁺ transcriptif it is represented in only a few percent of the TLC1 RNA population.

The inventors currently believe that the yeast telomerase RNA is notpoly-adenylated, but that the subset of TLC1 cDNAs with poly-A tractsthat the inventors isolated represent a by-product of the cDNAsynthesis. However, if the telomerase RNA is polyadenylated in S.cerevisiae, it may represent another level of control. For instance,genes involved in poly-A⁺ addition, degradation and message localizationhave been identified in yeast, and may be important in regulating TLC1activity (Muhlrad & Parker, 1992).

B. TLC1 RNA Expression and Cell-Cycle

A simple mechanism for limiting telomerase activity to a specific periodof the cell cycle, is to regulate the presence of the telomerase RNA.Therefore the steady-state levels of the TLC1 RNA through the cell cycleare determined. Methods are available to bring a culture of yeast cellsinto a synchronized progression through the cell cycle, or to arrest thecells at specific stages (Aparicio & Gottschling, 1994). For instance,MATa cells are arrested in late G1 with α-factor or a conditionalcell-cycle arrest mutation. Steady state RNA levels are then isolatedand analyzed. In addition, the cells are later released from the arrestand allowed to progress synchronously through the cycle, with RNAsamples being taken at various times after release. The cell cycleposition of the cell population is determined by examining cellmorphology and RNA levels of genes known to be cell-cycle regulated(e.g. CLN2 and SWI5). During the analysis of the TLC1 RNA, any changesin transcript length, particularly if a fraction of the RNA is modified,such as by poly-adenylation, is noted. A cell-cycle change may be theresult of cell-cycle regulated transcription or a post-transcriptionalevent such as RNA degradation.

C. Characterizing the TLC1 Gene

The inventors verified that the cDNA clones of TLC1 isolated areidentical to the genomic sequences. Thus, it does not appear that anymajor sequence modifications occur to the telomerase RNA aftertranscription, such as RNA splicing or editing (Moore et al., 1993;Bass, 1993), although the possibility of post-transcriptionalmodifications, such as methylation (Reddy & Busch, 1988), cannotpresently be ruled out.

The precise positioning of TLC1 within the genome, and the sequences ofthe gene's transcriptional control elements, was also determined by theinventors. The 3′ ends of TLC1 and CSG2 converge from oppositedirections. The CSG2 gene has a predicted ORF that terminates within 50bp of the 3′ end of the TLC1 cDNA sequences. On the opposite side ofTLC1, PDX3 has a divergent transcript with an ORF beginning ˜650 bp fromthe 5′ end of the TLC1 cDNA sequences. Analysis of this intervening 650bp, particularly in the region within 200 bp of the predicted TLC1 5′end, reveals matches or very near matches to TATA elements, GCN4 (Hillet al., 1986) and HOMOL1 (Rotenberg & Woolford, Jr., 1986) consensusbinding sites (both are transcriptional activators that bind to UpstreamActivating Sequences (UAS)), and to A block and B block sites(Geiduschek & Tocchini-Valentini, 1988, RNA Polymerase III controlelements). Thus at this point, TLC1 may be transcribed by either Pol IIor Pol III.

In order to determine which polymerase transcribes the gene, the steadystate level of the TLC1 message in strains containing a conditionaltemperature sensitive allele of either Pol II or Pol III is examined,after the cells have been shifted to a non-permissive temperature(Gudenus et al., 1988; Kolodziej & Young, 1991). This analysis, inconcert with 5′ deletion analysis, allows the RNA polymerase thattranscribes the gene to be determined. Sequences for two knowncell-cycle specific control elements, an Mlu I site or SWI4/SWI6consensus binding site (Nasmyth, 1993; Primig et al., 1992), are notpresent upstream of TLC1. Thus it is unlikely that TLC1 transcription isregulated by either of these cell-cycle dependent pathways.

The minimum extent of the sequences, both 5′ and 3′ of TLC1, that arerequired on a single-copy CEN plasmid to complement the chromosomal nullmutation tlc1::LEU2 are determined using, e.g., the plasmid PAZ1(obtained from Teresa Dunn, Beeler et al., 1994), which contains a 5.5kbp Sal I fragment that encodes all of CSG2 and TLC1, and most of PDX3.Using restriction enzymes and exonucleases the essential sequences aredetermined. The reduced size of the gene fragment will greatlyfacilitate further mutant analysis, and the 5′ deletion analysis willdetermine which UAS-promoter elements are essential for expression,thereby facilitating the creation of a conditional mutant with aheterologous UAS/promoter.

D. Constructing an Allele of TLC1 that is Regulated by a HeterologousPromoter

In order to facilitate in vivo studies on TLC1, a conditional allele ofthe gene is useful. A chimeric fusion of the TLC1 gene placed under theregulation of a heterologous promoter/UAS is contemplated. Based on datafrom the TLC1 DNA sequence, the 5′ end of the TLC1 RNA, and determiningwhat sequences at the 5′ end of the gene are essential for TLC1expression, the TLC1 upstream region is then replaced with the controlelements of the MET3 gene (Cherest et al., 1987). The MET3 promoter isrepressed in the presence of methionine and induced when methionine isabsent from the medium. MET3 transcriptional fusions to a number of RNAPol II transcribed genes have been described. The GAL1,10 UAS may alsobe used (Johnston & Davis, 1984). If TLC1 is transcribed by Pol III, thebacterial tetracycline repressor-operator system may be used to regulatethe TLC1 gene. A Pol III-transcribed gene has been shown to be regulatedby this system when the tetO operator sequence was introduced near the5′ end of the gene in S. cerevisiae (Dingermann et al., 1992).

The plasmid shuffle technique (Sikorski & Boeke, 1991) and conditionalalleles may also be used in place of heterologous promoters.

EXAMPLE XII The Role of TLC1 in Additional in vivo Processes

A. Telomerase RNA and Single-Strand TG₁₋₃ Tails

A single-strand TG₁₋₃ tail at the ends of yeast telomeres is transientlydetected late in S phase (Wellinger et al., 1993). This tail may be theresult of elongation of the 3′ strand at chromosome ends by telomeraseactivity. Tail formation in a tlc1 strain (Welinger et al., 1993) isthus examined. The TG₁₋₃ tails are detected by using a Southernhybridization method in which yeast DNA is never denatured, and thenhybridized with a C₁₋₃A probe. When the tails are ≧65 nucleotides long,the probe efficiently hybridizes to the single-strand of TG₁₋₃. Theanalysis is performed on cells that are synchronously progressingthrough S phase after release from an α-factor arrest (late G1), or acdc7 arrest (G1-S boundary). In a wild type (TLC1) cell the same resultsas previously observed are expected, but in a tlc1⁻strain, no taildetection is contemplated. However, if a single-strand tail is stillobserved in a tlc1⁻strain, then the tail is likely to be formed by atelomerase-independent mechanism. For instance, the tail may be formedby loss of terminal 5′ C₁₋₃A strand sequences, the result of acell-cycle controlled exonuclease activity. The tlc1 allele used in thisstudy is a conditional allele placed under a heterologous promoter.Alternatively, young (<50 generations old) haploid cells, the sporeproducts of a TLC1/tlc1 diploid strain, are used.

The role of EST1 in tail formation (Lundblad & Szostak, 1989) is alsoexamined. If tail formation is dependent upon both TLC1 and EST1, itlends support that EST1 is part of telomerase, or regulates itsactivity. Alternatively, if the tail is only dependent upon TLC1, itsuggests that EST1 may be important in another aspect of telomerereplication, perhaps in synthesis of the 5′ strand.

B. Telomerase RNA and Healing Broken Chromosome Ends

When a chromosome is broken, two non-telomeric DNA ends are generated;these ends are unstable. One mechanism for stabilizing ends is to ‘heal’them by the addition of telomeric sequences. Telomerase activity mayprovide a major mechanistic pathway for healing by adding telomere DNAde novo to the broken ends (Kramer & Haber, 1993; Harrington & Greider,1991). An alternative pathway, which has been documented in S.cerevisiae and Drosophila, utilizes recombinational mechanisms(Biessmann & Mason, 1992).

To test telomere healing, a Haber-based assay is used (Kramer & Haber,1993). In this system, a recognition sequence for the HO endonuclease islocated at a unique site in the genome of a diploid cell (on only one ofthe homologues), with markers genes on either side of it. The HOendonuclease is then conditionally expressed (it is under control of agalactose-dependent promoter) and results in cleavage of the singlehomologue. The strain is rad52⁻, which eliminates the major mitoticrecombination pathway in yeast, thus preventing repair of the brokenchromosome by gene conversion from the uncut homologue, or telomerehealing by the recombination pathway (Lundblad & Blackburn, 1993). Byselecting for cells that retain a marker centromere proximal to the cutsite, and loss of a marker telomere proximal to the cut, healedchromosomes are identified. A diploid cell is required in this system,because essential genes are lost distal to the cut site; these genefunctions are provided by the uncut homologue.

The inventors have designed a new genetic system, improved (FIG. 8) inseveral important ways: (1) The unique HO cleavage site is introduced atthe ADH4 locus into a haploid strain. ADH4 and the sequences distal toit are non-essential for haploid growth; thus, they may be lost withoutapparent consequence (Gottschling, 1990). The haploid nature of thestrain is of particular use in genetic identification and analyses ofrecessive mutations. (2) A short tract of TG₁₋₃ is placedcentromere-proximal of the HO cleavage site. This sequence serves as a‘seed’ for the healing event, thus increasing the probability that astable chromosome will be recovered. Correlative evidence from healedchromosomes in both yeast and humans indicate that normal occurrences ofsuch sequences at internal chromosomal loci are the major sites of denovo telomere addition (Kramer & Haber, 1993; Harrington & Greider,1991). (3) The LYS2 gene is located on the telomere-proximal side of theHO site, and HIS3 is located on the centromere-proximal side of theTG₁₋₃ sequence. The combination of these two genes provides a stronggenetic selection for the healing event. The loss of LYS2, and henceloss of the region distal to the cut site, is selected by growth onα-aminoadipate (α-AA) (Zaret & Sherman, 1985). Simultaneous selectionfor HIS3 (growth in the absence of histidine) ensures that sequencescentromere-proximal to the cleavage site are still present (Aparicio &Gottschling, 1994).

The strain contains one additional difference, the TLC1 gene is aconditional allele, under control of the MET3 promoter. Loss of TLC1function is accomplished by turning off the MET3 promoter (by theaddition of methionine to the media), thus allowing the requirement forTLC1 function in telomere healing to be tested.

It is expected that when HO endonuclease is expressed (by the presenceof galactose in the medium) in TLC1⁺ cells, the VII-L chromosome will becleaved at the HO site. Those cells that have formed a new telomere ator near the TG₁₋₃ ‘seed’ sequences, and have lost the distal LYS2 gene(presumably to nuclease degradation, inability to replicate, ormissegregation) 5 will be selected on −his +α-AA plates. The selectionwill be imposed on the cells several generations after HO cleavage. Thisis to avoid phenotypic lag during α-AA selection, due to the initialpresence of the LYS2 gene product.

It is expected that HO endonuclease cleavage will occur in nearly 100%of the cells in the population (FIG. 8), and that at least 1/1000 cellswill heal at the TG₁₋₃ ‘seed’ (Kramer & Haber, 1993). Those chromosomesthat do not heal at the ‘seed’ may form a new telomere at a morecentromere-proximal chromosomal position, or be completely lost. In theevent an essential gene or the entire chromosome is lost, the cell isinviable; if a new telomere is formed at a viable chromosomal location,the cell will be His⁻. To verify that the telomere has indeed healed asexpected, Southern analysis of the chromosome in this region isperformed. When the study is repeated in the absence of functional TLC1gene product, it is expected that no growth will be observed on −his+α-AA media. In fact, the process of HO cleavage in tlc1⁻ cells mayresult in complete inviability, as the lack of telomerase activity to‘heal’ the broken chromosome end may result in chromosome loss. Ifgrowth on −his +α-AA media is observed in tlc1 cells, it may be theresult of a loss of function mutation in the LYS2 gene in a small subsetof cells where cleavage did not occur (this is determined by Southernanalysis).

Of course, the original diploid system developed by Haber may be used inthis analysis. The assays used to characterize loss of TLC1 and EST1function in vivo, namely a decrease in telomere length and cellviability with increased age of a culture (in a rad52strain), can alsobe used for further analysis (Lundblad & Blackburn, 1989).

EXAMPLE XIII Genetic Dissection of TLC1 RNA

The telomerase RNA molecule is dissected to identify regions that areessential for telomerase activity and to define regions that interactwith other telomerase components. Two different genetic approaches areused. First, the technique that resulted in the original identificationof TLC1, namely, overexpression of TLC1 cDNAs to suppress telomericsilencing. Limited sequences within the RNA that are responsible for thesuppression are defined. These regions will interact with othertelomerase components and are useful in identifying these components.

Second, methods used to dissect small nuclear RNAs (snRNAs) and theirfunction in yeast (Parker, 1989: Guthrie & Patterson, 1988) are adapted.Here mutants of TLC1 are constructed and tested for in vivo functions,such as the ability to ‘heal’ broken chromosomes or form single-strandedtails late in S phase. Again, important regions of the RNA areidentified and used to isolate interacting components.

Methods to identify important regions of snRNAs include phylogeneticcomparisons of each type of RNA (e.g. U1, U2, etc.) (Miraglia et al.,1991; Ares, Jr., 1986; Ares, Jr., & Igel, 1990). Conserved sequences andsecondary structures in the RNA molecules of different species have beenanalyzed. Comparisons between telomerase RNAs from a variety of ciliateshave suggested conserved secondary structures, while little conservationat the primary sequence level is detected (Lingner et al., 1995; Romero& Blackburn, 1991). So far, conserved sequences or structures betweenthe 1.3 kb TLC1 RNA and the much smaller ciliate RNAs (the largest is200 nucleotides) have not been identified, but continued analyses mayyield useful information. While a similar size difference is seenbetween the long U2 snRNA from S. cerevisiae and the smaller U2's invertebrates, conserved primary sequences between U2 RNAs facilitatedstructural alignments that identified critical stems and loops in theRNA.

A. Minimal Sequence Elements in TLC1 RNA that Suppress TelomericSilencing

The same strains and expression vector used to identify TLC1 cDNAs areused to identify limited regions of the telomerase RNA that suppresstelomeric silencing. The full length telomerase RNA is examined todetermine whether it has the ability to suppress silencing at highlevels. While this molecule is expected to suppress very well, it ispossible that only truncated, non-functional telomerase RNAs have thisphenotype when overexpressed (a dominant-negative phenotype)(Herskowitz, 1987). Nonetheless, the full length RNA serves as thestarting point for creating 5′ and 3′ deletion derivatives, as well asderivatives that either delete internal segments or retain a singleinternal element. It is contemplated that relatively small regions ofthe RNA (perhaps 50 bp) that suppress silencing will be identified. Byreducing the size, an assured interaction of a single component and theRNA fragment is determined. This increases the likelihood of identifyingthe component.

It is believed that overexpression of TLC1 causes suppression oftelomeric silencing because the RNA titrates away a limited component inthe cell. To determine whether the limited component is part oftelomerase, part of the telomeric silencing machinery, or plays a rolein both complexes, each of the TLC1 overexpression derivatives aretested in other assays, e.g., in particular, the telomere ‘healing’assay that can be performed quantitatively. The derivatives that havethe strongest effect in reducing the frequency of healing are the bestcandidates for a telomerase-specific interaction.

This titration assay is contemplated for use in identifying telomeraseRNA structures that are conserved between species. For instance, thetelomerase RNA from a ciliate such as Oxytricha may act to suppresstelomeric silencing when expressed at high levels, if the Oxytricha RNAis able to interact with a conserved telomerase component in yeast. Ifsuch structural conservation occurs, this assay is then useful forisolating telomerase RNAs from species in which the RNA has not yet beenisolated, such as from humans.

B. Creating TLC1 Mutants

A second method to identify important regions of the TLC1 RNA that mayinteract with other telomerase components involves making loss offunction mutations in the RNA, excluding the template region. With anRNA as large as the TLC1 transcript, such mutations are relatively easyto isolate and, indeed, specific regions of the RNA will be mutated.Either site-directed or limited random mutations to regions of TLC1 forwhich there is evidence of a conserved secondary structure, or forinteraction with other telomerase components, are thus made. Suchregions of TLC1 RNA include those that can suppress telomeric silencingwhen overexpressed, or contain predicted secondary structures that areconserved between TLC1 RNA and telomerase RNAs from other species. Thesemutations may define dominant, semi-dominant, or recessive alleles ofTLC1.

Screening for recessive mutations is first advised because they can bemore easily manipulated. Conditional alleles that are sensitive totemperature or moderate structural perturbations, such as lowconcentrations of formamide or D₂O, typically are of greater utilityidentifying interacting proteins than mutations which are complete lossof function (Huffaker et al., 1987; Bartel & Varshavsky, 1988). Thesealleles are isolated by a “plasmid shuffle” scheme (Sikorski & Boeke,1991): One centromere plasmid that contains both the URA3 and wild typeTLC1 genes is introduced into a strain deleted for the normalchromosomal copy of TLC1 and containing the required genotype for thetelomere ‘healing’ assay. A second centromere plasmid, with a differentselectable marker such as TRP1, carries a mutated TLC1 gene. Themutagenized plasmid(s) are then transformed in the appropriate yeaststrain, and a screen for conditional alleles of TLC1 is carried out.

Mutants of interest are those that allow a transformant to “heal”telomeres on −trp +FOA medium (losing the wild type TLC1-URA3 plasmidand retaining the mutant version, which still functions) only when grownat the permissive temperature. At the nonpermissive temperature, suchstrains are unable to heal telomeres in the presence of FOA becausehealing is dependent on wild-type TLC1 RNA (growth on FOA can only occurin the absence of the URA3 gene product (Boeke et al., 1987). Therelative ability of these alleles to function in the healing assay isquantified by determining the frequency of chromosome healing. Thequantitative analysis is useful in classifying the alleles and isolatingeither suppressors or enhancers. The TLC1 alleles are also screened inother assays, such as formation of single-strand tails, to determine ifthere are mechanistic differences between the alleles.

EXAMPLE XIV Isolation of Genes that Interact with TLC1

Based on the two types of TLC1 derivatives created, genetic screens arecarried out to isolate genes whose products interact with the telomeraseRNA.

A. Genes that Re-Establish Telomeric Silencing when TLC1 RNAs areOverexpressed

This approach is based on the model that when parts of the TLC1 RNA areoverexpressed, they interact with a limiting telomerase component toform a non-functional complex. This reduces the level of telomeraseactivity in the cell, causing reduced telomere length, and the reductionin telomere length decreases the frequency with which telomericsilencing complexes are assembled. Thus, if the concentration of thecomponent is increased such that it is no longer limiting, telomeresbecome longer and telomeric silencing is re-established.

In this approach, the small TLC1 fragment(s) are expressed at a levelthat is only slightly higher than necessary for suppression of telomericsilencing. This way, only a small amount of the limiting component isneeded to re-establish silencing. The threshold concentration for theGAL1-TLC1 RNA fragment to suppress telomeric silencing is determined bydecreasing the concentration of galactose in the medium; expression fromthis promoter is modulated by galactose concentration. The actualthreshold is determined by measuring steady-state RNA levels as afunction of telomeric silencing. If a threshold concentration isachieved, the construct is integrated into the genome to help keep theTLC1 RNA fragment level constant.

Once a suitable concentration of the TLC1 fragment is established, thegene encoding the limiting telomerase component is isolated byidentifying an overexpression plasmid that, when introduced into thisstrain, re-establishes silencing. Yeast plasmid libraries that may beused include high copy genomic libraries and cDNA libraries, e.g.,driven by the ADH1 promoter (obtainable from S. Elledge).

Candidate plasmids are isolated by a reversal of the selection procedureused to originally identify TLC1. The starting strain contains theconstruct that expresses the TLC1 fragment at high levels in addition tohaving two telomeres marked, one with ADE2 the other with URA3. In thisstrain, telomeric silencing is suppressed by the expression of the TLC1fragment; the cells are sensitive to growth on FOA (URA3⁺), and arewhite (ADE2⁺). When silencing is re-established, the cells are able togrow on FOA (FOA^(R); ura3)and form red/white sectored colonies(red=ade2). After the library plasmids are transformed into the strain,re-establishment of silencing is selected/screened by growth ofred/white colonies on FOA. In addition to components that interact withTLC1, some plasmids may be isolated that affect the expression level ofthe GAL1-driven TLC1 fragment. This class is identified by examining thesteady state level of the TLC1 RNA fragment. This class may representgenes that negatively regulate GAL1 transcription, or genes thatregulate RNA stability.

However, it is also possible that more than one gene product may belimiting in the titration; for example the RNA fragment may be bound bya dimeric complex, with the two components at low concentration in thecell. The limiting factor may be lethal to yeast cells at highconcentration, “fouling” an essential cellular function. Therefore, theTLC1 RNA fragment may be used to probe a lgtll yeast cDNA expressionlibrary. An in vitro synthesized ³²P-labeled RNA, identical to thatdefined in vivo, is used to probe a set of filters containing phageplaques. Those plaques that contain a cDNA expressing a TLC1 interactingprotein are isolated by virtue of their ability to bind the radioactiveprobe.

For those plasmids that are candidates for encoding a TLC1 interactingcomponent, the DNA necessary for the effect is determined and subjectedto sequence analysis. Putative genes are then subjected to the sameanalyses used to identify TLC1 as a telomerase component. That is,examining telomere length and cell viability in a strain with a nullmutation of the gene and the gene is characterized in biochemicalanalyses.

B. Modifiers of Conditional TLC1 Mutations

A more classical approach for identifying components that interact withtelomerase RNA may be used, e.g., by isolating mutations that enhance orsuppress the phenotypes of conditional alleles of TLC1 (as createdabove). This genetic approach has been successfully utilized inidentifying components from many complex biological systems, includingproteins that interact with snRNAs involved in splicing (Parker, 1989;Guthrie & Patterson, 1988).

Mutations that suppress the defect of telomere healing in conditionalalleles of tlc1 under non-permissive conditions are isolated. Thestarting strain includes the “healing” set-up in addition to a specificconditional allele of tlc1. At non-permissive temperatures, this strainis unable to grow on −his+α-AA medium after HO endonuclease induction(or as noted, induction may be lethal), the result of a defect intelomere healing. This strain is then mutagenized and mutations thatpermit ‘healing’ to occur (growth on −his+α-AA medium) are isolated. Thestrains containing the suppressors are back-crossed to a parent strain(congenic with the starting strain except the opposite mating type) andthe resulting diploid is sporulated and tetrads dissected. After severalsuch backcrosses to isolate the suppressor mutation from other mutationsthat may have been introduced during mutagenesis, the suppressionphenotype is checked to see that it segregates 2:2. Once isolated, thesuppressor is crossed to strains containing other tlc1 alleles todetermine if it acts specifically on the allele from which it wasisolated. If there is allele specificity, then the suppressor mutationinteracts with TLC1 RNA in vivo (Huffaker et al., 1987). If not, thereis still a high probability that the suppressor interacts with TLC1.Possible suppressor linkage to tlc1 mutations are also determined.

The strategy to isolate the gene encoding the suppressor depends onwhether the mutation is dominant, semi-dominant, or recessive, andwhether the mutation has additional phenotypes that may be followed.Dominant mutations are currently preferred. A centromere-based genomicDNA library made from the strain containing the suppressor is used totransform the non-mutated ‘healing’, tlc1 strain. Those plasmids thatpermit telomere healing, as described above, and do not encode the wildtype TLC1 gene, carry the suppressor gene.

An alternative method is to isolate genes that suppress a mutation whenin high dosage (Bender & Pringle, 1991). Suppressors can thus bescreened for by transforming with a high copy genomic library andisolating plasmids that suppress the telomere healing defect.

Enhancers of the conditional TLC1 alleles grown at permissive orsemi-permissive temperatures may also be isolated, as has beensuccessful in identifying interacting components within many biologicalprocesses of yeast (Frank et al., 1994).

C. Continued Characterization of the STR Genes

Two of these genes, STR5 and STR6 have a much stronger affect ontelomeric silencing than on silencing at HML though not as strikingly asTLC1 does (Example X; FIG. 7A, FIG. 7B). If null mutations of thesegenes have similar effects on telomeres as those seen in tlc1⁻-strains,they are excellent candidates for being components of telomerase.

EXAMPLE XV Biochemical Approaches to Telomerase

Telomere DNA binding proteins from Oxytricha have been isolated andcharacterized (Gottschling & Zakian, 1986). In vitro transcriptionassays with yeast extracts and proteins have also yielded low abundancetranscription factors (Parthun & Jaehning, 1992). Therefore, proteinelements of telomerase are isolatable.

A. Biochemical Characterization of the Ribonucleoprotein ComplexAssociated with TLC1 RNA

To examine the physical association of genes with TLC1, procedures usedto isolate small nuclear ribonucleoprotein particles (snRNPs) areadapted (Luhrmann, 1988). The approximate steady state concentration ofTLC1 RNA within cells is first determined, by comparing the amount ofthe RNA isolated from a given number of cells with a dilution series ofin vitro transcribed TLC1 RNA. The information obtained from thisanalysis indicates how much telomerase activity and associated proteinis in a cell, and serves as an indicator for enrichment of the TLC1ribonucleoprotein complex during fractionation procedures.

The first fractionation step separates the nucleus and the cytoplasm,using procedures described for other ribonucleoprotein complexes inyeast (Hopper et al., 1990). It is expected that all TLC1 RNA will belocalized to the nucleus, however a cytoplasmic location is notexcluded. In the event TLC1 RNA is in the cytoplasm, the fractionationis performed on cells that are arrested at various points in the cellcycle (with pheromone, CDC mutations, or chemicals).

Next, with RNA in the nucleus, a nuclear extract is made andfractionated to give a TLC1 RNA associated particle e.g., by acombination of gradient centrifugation methods (equilibrium andsedimentation velocity), column chromatography steps, includinggel-filtration, ion-exchange, hydrophobic/ion-exchange, and agarosebeads linked to dyes and other ligands, and gel electrophoresis(Luhrmann, 1988). In addition, buffer and ion conditions are carefullymonitored as they can affect the stability of the particle (Roth et al.,1991). An affinity column for TLC1 RNA is also contemplated, e.g., as iscreated by synthesizing a biotinylated DNA oligonucleotide that iscomplementary to the RNA's template sequence. The oligonucleotide, whichwill hybridize to the RNA in the particle, is then tethered tostreptavidin beads (Kijas et al., 1994).

As a first use of the fractionation, the fate of EST1 may be followedusing protein anti-EST1 antibodies (available from Dr. V. Lundblad) asgenetic evidence suggests that it may be part of or regulate telomerase(Lundblad & Szostak, 1989). It is contemplated that extracts from twodifferent mutants that both have ‘defective’ particles will be combinedto generate a fully assembled particle, thus allowing insights into theparticle's biogenesis.

Reagents, such as antibodies, to proteins identified in the geneticscreens are also contemplated.

B. In vitro Assay for Telomerase Activity from S. cerevisiae

Telomerase activity has been biochemically identified from severalciliates and vertebrates, including human cells. However, prior to thepresent invention, telomerase activity had not been biochemicallydetected in S. cerevisiae. Now assays are available, based partly onthose previously described (Greider & Blackburn, 1985; Mantell &Greider, 1994; Prowse et al., 1993; Autexier & Greider, 1994; Greider &Blackburn, 1987), in which a DNA oligonucleotide substrate, representingthe 3′ G-rich telomere tail, is incubated in extracts with ³²P-labeleddNTP's (typically dGTP or dTTP). The products of telomerase elongationon the input oligonucleotide substrate are then detected by gelelectrophoresis and autoradiography.

Identifying the TLC1 RNA and its sequence will likely assist inisolating the activity. To identify telomerase activity in yeast, bufferconditions that have been successful in other systems are used anddamaging nucleases are removed. In addition, extracts from strains thatare deficient in several of the major proteases (Jones, 1991), and usecocktails of protease inhibitors that have been successfully used for invitro transcription (Parthun & Jaehning, 1992) are employed.

A series of substrates, ones that are perfectly complementary to thetemplate, are truncated on their 3′ end by one or a few nucleotides, orare simply alternating tracts of (GT)_(n) are used in isolation studies,and very short oligonucleotide products are also analyzed. As telomeraseactivity in yeast may be very tightly regulated, and limited to only abrief period of the cell cycle, (Wellinger et al., 1993), extracts fromcells isolated in a synchronous population late in S phase are also tobe used.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

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What is claimed is:
 1. A method of using a DNA segment that comprises anisolated gene associated with non-ciliate telomerase, wherein said DNAsegment is characterized as encoding a polypeptide that includes acontiguous amino acid sequence of at least about 17 amino acids from SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, oris characterized as specifically hybridizing to the nucleic acid segmentof SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:19, SEQ ID NO:31 or SEQ IDNO:23, or the complement thereof, the method comprising the steps of:(a) preparing a recombinant vector in which a non-ciliatetelomerase-associated gene is positioned under the control of apromoter; (b) introducing said recombinant vector into a recombinanthost cell; (c) culturing the recombinant host cell under conditionseffective to allow expression of the telomerase-associated gene; and (d)collecting the expressed gene product.
 2. A method for modifying thetelomerase activity of a cell, comprising contacting atelomerase-containing cell with an amount of a composition effective tomodify telomerase activity, said composition comprising: (a) an isolatedRNA segment of from 25 to about 1,500 nucleotides in length thatcomprises a non-ciliate telomerase RNA template, the RNA segmentspecifically hybridizing to the nucleic acid segment of SEQ ID NO:1 orthe complement thereof under high stringency hybridization conditions;or (b) an isolated telomerase-associated protein or polypeptide thatincludes a contiguous amino acid sequence of at least about twelve aminoacids from SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQID NO:24, and assaying said cell for telomerase activity.
 3. The methodof claim 2, wherein said composition comprises: (a) a nucleic acidsegment that includes the DNA sequence of SEQ ID NO: 1; or (b) a nucleicacid segment that includes the contiguous DNA sequence from position 54to position 1799 of SEQ ID NO:29, the contiguous DNA sequence fromposition 78 to position 1094 of SEQ ID NO:30, the contiguous DNAsequence from position 2 to position 2368 of SEQ ID NO: 19, thecontiguous DNA sequence from position 55 to position 699 of SEQ IDNO:31, or the contiguous DNA sequence from position 3 to position 1955of SEQ ID NO:23.
 4. The method of claim 2, wherein saidtelomerase-containing cell is a human cell.
 5. The method of claim 2,wherein said telomerase-containing cell is a sperm cell.
 6. The methodof claim 2, wherein said telomerase-containing cell is an egg cell. 7.The method of claim 2, wherein said telomerase-containing cell is atumor cell.
 8. The method of claim 2, wherein said telomerase-containingcell is a pathogenic cell.
 9. The method of claim 2, wherein saidtelomerase-containing cell is located within an animal and apharmaceutically acceptable formulation of said composition isadministered to said animal.
 10. A method for modifying the viability acell with increased age, comprising contacting a telomerase-containingcell with an amount of a composition effective to modify telomeraseactivity, said composition comprising: (a) an isolated RNA segment offrom 25 to about 1,500 nucleotides in length that comprises anon-ciliate telomerase RNA template, the RNA segment specificallyhybridizing to the nucleic acid segment of SEQ ID NO:1 or the complementthereof under high stringency hybridization conditions; or (b) anisolated telomerase-associated protein or polypeptide that includes acontiguous amino acid sequence of at least about twelve amino acids fromSEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.